Is There a Third Photoreceptor Involved in the Control of Chloroplast Movements in Mougeotia?

The photometric method was used to test a possibility proposed recently that a new photoreceptor with maximum activity at 620 nm is involved in mediating chloroplast rotation in Mougeotia (Z Lechowski, J Bialczyk [1988] Plant Physiol 88: 189-193). The hypothesis was tested under conditions of continuous dichromatic unilateral or mutually perpendicular irradiation with red light of wavelengths 620 or 660 (680) nanometers and far-red. When the red light was polarized parallel to the long cell axis, chloroplast response could be monitored by changing the direction of far-red irradiation. The level of the response obtained with red and far-red applied from the same direction depended on far-red intensity: at higher fluence rates the maximum response was shifted to longer wavelengths of red light. A high fluence rate of far-red inhibited the response. The absorption coefficients of Mougeotia chloroplasts were measured for the studied wave-lengths using the microphotometric method. Possible impact of absorption by the chloroplast on photoreception has been discussed. Current and previous results can be interpreted in terms of phytochrome action and do not support the involvement of the hypothetical 620 nanometer photoreceptor.     

Interaction between Green and Far-Red Light on the Low Fluence Rate Chloroplast Orientation in Mougeotia

Continuous irradiation of Mougeotia with linearly polarized green light (550 nanometers, 0.2 watt per square meter) induces a change in the orientation of its chloroplast from profile to face position, if the electrical vector of the green light is vibrating normal to the cell axis. This change is complete within 25 minutes of the onset of irradiation. In contrast, if the electrical vector of the green light is parallel to the cell axis, no chloroplast reorientation is induced, even with a fluence rate as high as 3 watts per square meter. Furthermore, unpolarized far-red light (727 nanometers, 2 watts per square meter) given alone has no effect on chloroplast reorientation. Simultaneous and continuous irradiation with polarized green light, regardless of its plane of polarization, together with unpolarized far-red light, however, does lead to chloroplast reorientation. These data indicate that, in addition to the red-absorbing form of phytochrome, there exists in Mougeotia another sensory pigment absorbing green light.     

Phytochrome Activation of K+ Channels and Chloroplast Rotation in Mougeotia: the Escape Times

Red light mediates chloroplast movement and increased activityof calcium-activated potassium channels on the plasma membraneof the alga Mougeotia sp. (UTEX LB 734). When activation ismediated by phytochrome, a far-red light irradiation given sometime after the red light irradiation will reverse the effectof the red light, due to phytochrome photoreversibility. Wecharacterized the escape times (time required for loss of photoreversibility)for these two processes to compare the transduction pathwaysinvolved in chloroplast rotation and channel activation. Theescape time for chloroplast rotation was 2.5 min after red lightirradiation (red and far-red light irradiations were 30 s).For channel activation, shorter red and far-red light irradiations(10 s) had to be used to obtain an escape time of 20 s. Thedifference in the escape times suggests that there is relativelyrapid divergence in the transduction pathways leading from phytochromeactivation (only one molecular species of phytochrome is foundin Mougeotia) to each of the two responses in the same cellularsystem. Because channel activation occurs 2–4 min afterirradiation while the escape time is 20 s, it is unlikely thatphytochrome acts directly on the channel. (Received September 26, 1995; Accepted December 28, 1995)     

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

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

Blue-light-induced chloroplast orientation in Mougeotia. Evidence for a separate sensor pigment besides phytochrome

Chloroplast orientation in the green alga Mougeotia has been induced by unidirectional red or blue light, given continuously during one hour. In addition, part of the preparations obtained scattered strong far-red light simultaneously with the orienting light. This far-red light completely abolished the response to red light, consistent with phytochrome as the sensor pigment for orientation in Mougeotia. In blue light, however, the response was completely insensitive to far-red light, thus pointing to a different sensor pigment in the shortwavelength region.Abbreviation P_fr far-red-absorbing form of phytochrome     

Die Chloroplastendrehung beiMougeotia

Summary Mougeotia cells with chloroplasts oriented in profile have been irradiated with small spots of monochromatic red polarized light in order to induce chloroplast movement.In these experiments, four factors have been varied: 1. the orientation of the vibration plane of the light in relation to the cell axis, 2. the localization of the spot, i. e. irradiation of the chloroplast or the cytoplasm, 3. the spot size, and 4. the duration of the irradiation.As a result of our experiments, we conclude that the photoreceptor molecules responsible for the light-induced chloroplast movement are localized in the cytoplasm.As the photoreceptor of this reaction is the well known phytochromesystem, we may assume that also in other plants the phytochrome is localized in the cytoplasm rather than in the chloroplast.

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Reversion from light-induced inhibition of seed germination by respiratory inhibitors

Treating of the dark-imbibed lettuce (Lactuca sativa L.) seeds prior to light irradiation with 1 millimolar KCN or NaN₃ in the dark for 3 hours prevented blue light and far-red light-induced inhibitions of phytochrome-mediated germination. Similarly, salicylhydroxamic acid (SHAM) at 10 millimolar counteracted the blue and far-red light inhibitions, the combined application of KCN and SHAM being more effective than KCN or SHAM alone in some experiments. These respiratory inhibitors slightly inhibited phytochrome-mediated lettuce seed germination. These results indicate that both CN-sensitive, conventional cytochrome oxidase and CN-resistant (SHAM-sensitive), alternative respiration may be involved in the light inhibition or that an appropriate balance of both may be necessary for the light inhibition.     

Chloroplast movement in Mougeotia induced by blue light pulses

The profile-to-face chloroplast movement in the green alga Mougeotia has been induced by strong blue and near-ultraviolet light pulses (6 J m^-2). Simultaneously, strong red or far-red light (10 W m^-2) was applied perpendicularly to the inducing beam. The response was measured photometrically. Against the far-red background the reciprocity law was found to hold for pulse durations varying two orders of magnitude. The action spectrum exhibited a maximum near 450 nm and a distinct increase in near-ultraviolet. The time-course and the spectral dependence of pulse responses of chloroplasts in Mougeotia were similar to those recorded for other plants which are sensitive only to blue. This points to an alternative sensor system active in the short-wavelength region in addition to the phytochrome system.Abbreviations FR far-red light - Pr red absorbing form of phytochrome - Pfr far-red absorbing form of phytochrome - R red light This paper is dedicated to the memory of Professor Jan Zurzycki     

Phytochrome-mediated branch formation in protonemata of the mossCeratodon purpureus

We have analyzed light induction of side-branch formation and chloroplast re-arrangement in protonemata of the mossCeratodon purpureus. After 12 hr of dark adaptation, the rate of branch formation was as low as 5%. A red light treatment induced formation of side branches up to 75% of the dark-adapted protonema. The frequency of light induced branch formation differed between cells of different ages, the highest frequency being found in the 5th cell, the most distal cell studied from the apex. We examined the effect of polarized light given parallel to the direction of filament growth. The position of branching within the cell depended on the vibration plane of polarized red light. Branch formation was highest when the electric vector of polarized light vibrates parallel to the cell surface and is fluence rate dependent. The positional effect of polarized red light could be nullified to some extent by simultaneous irradiation with polarized far-red light. An aphototropic mutant,ptr116, shows characteristics of deficiency in biosynthesis of the phytochrome chromophore and exhibits no red-light induced branch formation. Biliverdin, a precursor of the phytochrome chromophore, rescued the red-light induced branching when added to the medium, supporting the conclusion that phytochrome acts as photoreceptor for red light induced branch formation. The light effect on chloroplast re-arrangement was also analyzed in this study. We found that polarized blue light induced chloroplast re-arrangement in wild-type cells, whereas polarized red light was inactive. This result suggests that chloroplast re-arrangement is only controlled by a blue light photoreceptor, not by phytochrome inCeratodon.     

Effects of Colchicine and Light on Cell Form in Fern Gametophytes. Implications for a Mechanism of Light-induced Cell Elongation

Spores of the fern, Onoclea sensihilis L., suffer a disruption of normal development when they are cultured on media containing colchicine. Cell division is inhibited, and the spores develop into giant spherical cells under continuous white fluorescent light. In darkness only slight cell expansion occurs. Spherical cell expansion in the light requires continuous irradiation. Photosynthesis does not seem to be involved, since variations in light intensity do not affect the final cell diameter; the addition of sucrose to the medium does not permit cell expansion in darkness; and the inhibitor DCMU does not block the light-induced cell expansion. Continuous irradiation of colchicine-treated spores with blue, red or far-red light produces different patterns of cell expansion. Blue light permits spherical growth, similar to that found under white light, whereas red and far-red light promote the reestablishment of polarized filamentous growth. Although ethylene is unable to induce polarized cell expansion in colchicine-treated spores in darkness or white and blue light, it enhances filamentous growth which already is established by red or far-red irradiation. Both red and far-red light increase the elongation of normal filaments (untreated with colchicine) above that of dark-grown plants, but under all 3 conditions the rates of volume growth are identical. Light, however, does cause a decrease in the cell diameters of irradiated filaments. These data are used to construct an hypothesis to explain the promotion of cell elongation in fern protonemata by red and far-red light. The model proposes light-mediated changes in microtubular orientation and cell wall structure which lead to restriction of lateral cell expansion and enhanced elongation growth.     

Kinetics of the dichroic reorientation of phytochrome during photoconversion inMougeotia

In the green algaMougeotia, the dichroic orientation of the red-absorbing form of phytochrome (P_r) is parallel of the cell surface, whereas the far-red-absorbing form (P_fr) is oriented normal to it. The time course of the change from parallel to normal was investigated by double-flash irradiation with polarized red and far-red light. The results obtained by two different methods indicate that most of the phytochrome intermediates existing in the first 5 ms after the inducing red flash are still oriented parallel to the cell surface, similar to P_r. At increasing intervals between the red and the far-red flashes, more and more phytochrome molecules turn their transition moments to the P_fr orientation. This reaction is finished after approximately 30 ms. We conclude that the change in dichroic orientation of the phytochrome molecules inMougeotia occurs during the last relaxation steps of the intermediates on the way from P_r to P_fr. It cannot be decided yet, whether the first surface-normal phytochrome species is an intermediate or P_fr itself.Abbreviations P_r red-absorbing form of phytochrome - P_fr far-red-absorbing form of phytochrome A preliminary report of this work was presented at the European Symposium on Photomorphogenesis, University of Reading, UK (Kraml et al. 1982)     

Diatom Phytochromes Reveal the Existence of Far-Red-Light-Based Sensing in the Ocean

The absorption of visible light in aquatic environments has led to the common assumption that aquatic organisms sense and adapt to penetrative blue/green light wavelengths but show little or no response to the more attenuated red/far-red wavelengths. Here, we show that two marine diatom species, Phaeodactylum tricornutum and Thalassiosira pseudonana, possess a bona fide red/far-red light sensing phytochrome (DPH) that uses biliverdin as a chromophore and displays accentuated red-shifted absorbance peaks compared with other characterized plant and algal phytochromes. Exposure to both red and far-red light causes changes in gene expression in P. tricornutum, and the responses to far-red light disappear in DPH knockout cells, demonstrating that P. tricornutum DPH mediates far-red light signaling. The identification of DPH genes in diverse diatom species widely distributed along the water column further emphasizes the ecological significance of far-red light sensing, raising questions about the sources of far-red light. Our analyses indicate that, although far-red wavelengths from sunlight are only detectable at the ocean surface, chlorophyll fluorescence and Raman scattering can generate red/far-red photons in deeper layers. This study opens up novel perspectives on phytochrome-mediated far-red light signaling in the ocean and on the light sensing and adaptive capabilities of marine phototrophs.     

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

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

Brief irradiation with red or blue light induces orientational movement of chloroplasts in dark-adapted prothallial cells of the fernAdiantum

Orientational movement of chloroplasts was induced by a brief irradiation with red light (R) or blue light (B) in dark-adapted prothallial cells ofAdiantum, whose chloroplasts had gathered along the cell dividing wall (i.e., the anticlinal wall). When the whole dark-adapted prothallia were irradiated from a horizontal direction (i.e., from their lobes) with horizontally vibrating polarized R (H pol. R) for 10 or 3 min, the chloroplast left the anticlinal walls and spread over the prothallial surface (i.e., the periclinal walls) within 1–2 hr after the onset of irradiation, returning to the anticlinal wall (dark-position) within 10 hr. However, vertically vibrating polarized R (V pol. R) for 10 min did not induce the movement towards periclinal walls. The R effect was cancelled by non-polarized far-red light (FR) irradiation just after the R irradiation. Irradiation with H pol. B for 10 or 3 min but not with V pol. B could also induce a similar movement of chloroplasts, although the chloroplasts returned within 4 hr. The effect of H pol. B, however, was not cancelled by the subsequent FR irradiation. When a part of the dark-adapted cell at the prothallial surface was irradiated from above with a microbeam of R or B for 1 min, chloroplasts of the cell in the dark-position moved towards the irradiated locus in subsequent darkness. However, in the neighboring cells, orientational movement was not induced by either R or B microbeams. These results show that in dark-adapted prothallial cells, both brief irradiation with R and B can induce chloroplast photo-orientation and that the photoreceptors are phytochrome and blue light-absorbing pigment, respectively. It is also clear that effects of both R and B irradiation do not transfer to neighboring cells.     

Photophysiology of Kalanchoë Seed Germination

The effects of irradiations with different proportions of red/farred light and of gibberellic acid on the phytochrome-mediated seed germination of Kalanchoë blossfeldiana cv. Feuerblüte, were studied. The seed coat transmits much more red than far-red light, and therefore the energy ratio between 660 nm and 730 nm is given only for the transmitted light. Decreasing this ratio from 65 to 1.0 caused only a very slight inhibition. If this ratio is further lowered to 0.64, a 10 min terminal irradiation after a 3-h white light photoperiod is inhibitory, but a 12-h photoperiod or continuous irradiation is not. If the ratio is decreased to 0.44 or 0.31, a 12-h photoperiod is now also inhibitory, although continuous irradiation and 10 min terminal irradiation are still more inhibitory. These results are discussed in terms of phytochrome phototransformations. Although gibberellic acid is unable to cause any germination in complete darkness, it can result in a very high germination percentage, if combined with treatments which by themselves do not induce any germination such as continuous far-red, terminal far-red after short photoperiods, or very short photoperiods at 25°C. These results point to a strong synergism between gibberellic acid and the so-called stabilized form of phytochrome, P*_FR.     

Short-term reactions of phytochrome in Mougeotia?

Summary Phytochrome controlled chloroplast movement in Mougeotia is induced by flashes of polarized red light. Two subsequent flashes, separated by a dark interval of a few seconds, are much more effective than two simultaneous flashes; a maximal cumulative effect is reached if the duration of the dark interval is 30 ms or longer. We propose two light reactions in series, separated by a very fast dark reaction. Preliminary evidence is given that the energy requirement for these light reactions is different. It is suggested that the two reactions are related in some way to free and bound phytochrome.Dedicated to Prof. Dr. E. Bünning on the occasion of his seventieth birthday.     

Phytochrome Control of the Development of Ascorbate Oxidase Activity in Mustard (Sinapis alba L.) Cotyledons

The activity of ascorbate oxidase (AOX) in mustard (Sinapis alba L.) cotyledons was markedly increased by irradiation with continuous far-red light. The involvement of phytochrome in this light-mediated response was demonstrated by red/far-red reversibility experiments. To determine immunochemically the contents of AOX in cotyledons, the antibody against the enzyme was raised in a rabbit. However, the antiserum was not monospecific to AOX; it also recognized glycoproteins. To remove antibodies that are specific to a carbohydrate moiety of glycoproteins, the anti-AOX antiserum was applied to a horseradish peroxidase-conjugated Sepharose column. By using the antibodies that were not retained in the column, the changes in the content of AOX were followed. Western immunoblot profiles revealed that the content of AOX protein in cotyledons notably increased after continuous far-red light treatment. Pulse-labeling experiments indicated that the synthesis of AOX protein occurred in the cotyledons. These results are in good agreement with the hypothesis that phytochrome-mediated increase in AOX activity is accompanied by the synthesis of the enzyme.     

Intracellular chloroplast photorelocation in the moss Physcomitrella patens is mediated by phytochrome as well as by a blue-light receptor

 The light-induced intracellular relocation of chloroplasts was examined in red-light-grown protonemal cells of the moss Physcomitrella patens. When irradiated with polarized red or blue light, chloroplast distribution in the cell depended upon the direction of the electrical vector (E-vector) in both light qualities. When the E-vector was parallel to the cross-wall (i.e. perpendicular to the protonemal axis), chloroplasts accumulated along the cross-wall; however, no accumulation along the cross-wall was observed when the E-vector was perpendicular to it (i.e. parallel to the protonemal axis). When a part of the cell was irradiated with a microbeam of red or blue light, chloroplasts accumulated at or avoided the illumination point depending on the fluence rate used. Red light of 0.1–18 W m⁻² and blue light of 0.01–85.5 W m⁻² induced an accumulation response (low-fluence-rate response; LFR), while an avoidance response (high-fluence-rate response; HFR) was induced by red light of 60 W m⁻² or higher and by blue light of 285 W m⁻². The red-light-induced LFR and HFR were nullified by a simultaneous background irradiation of far-red light, whereas the blue-light-induced LFR and HFR were not affected at all by this treatment. These results show, for the first time, that dichroic phytochrome, as well as the dichroic blue-light receptor, is involved in the chloroplast relocation movement in these bryophyte cells. Further, the phytochrome-mediated responses but not the blue-light responses were revealed to be lost when red-light-grown cells were cultured under white light for 2 d. Received: 7 September 1999 / Accepted: 15 October 1999     

EFFECTS OF VISIBLE AND ULTRAVIOLET RADIATION ON THE GERMINATION OF PHACELIA TANACETIFOLIA

Schulz , Sister M. Richardis , O.P., and Richard M. Klein . (N. Y. Bot. Gard., N. Y., N. Y.) Effects of visible and ultraviolet radiation on the germination of Phacelia tanacetifolia. Amer. Jour. Bot. 50(5): 430–434. Illus. 1963.—Germination of Phacelia tanacetifolia was suppressed by exposure to white light increasing with intensity and length of illumination. The light effect decayed during 24 hr of darkness. Seeds were most sensitive to the suppressive effects of light 13–17 hr after the beginning of imbibition. Light suppression was caused by a photocatalytic reaction. Wavelengths causing the suppression lie in the far-red, red, blue, near-ultraviolet and far-ultraviolet regions of the spectrum. At equal energies, blue light was less effective than far-red, red or ultraviolet radiation. There was no evidence for the existence of the phytochrome system. Simultaneous irradiation with red and blue light or simultaneous irradiation with red and far-red induced a synergistic repression of germination. The presentation of different wavelengths in various sequential patterns markedly altered the germination response. An interaction between elevated temperatures and visible radiation affecting germination response was also noted.