Phytochrome of the green alga Mougeotia: cDNA sequence, autoregulation and phylogenetic position

A cDNA clone encoding phytochrome (apoprotein) of the zygnematophycean green alga Mougeotia scalaris has been isolated and sequenced. The clone consisted of 3372 bp, encoded 1124 amino acids, and showed strain-specific nucleotide exchanges for M. scalaris, originating from different habitats. No indication was found of multiple phytochrome genes in Mougeotia. The 5 non-coding region of the Mougeotia PHY cDNA harbours a striking stem-loop structure. Homologies with higher-plant phytochromes were 52–53% for PHYA and 57–59% for PHYB. Highest homology scores were found with lower-plant phytochromes, for example 67% for Selaginella (Lycopodiopsida), 64% for Physcomitrella (Bryopsida) and 73% for Mesotaenium (Zygnematophyceae). In an unrooted phylogenetic tree, the position of Mougeotia PHY appeared most distant to all other known PHYs. The amino acids Gly-Val in the chromophore-binding domain (-Arg-Gly-Val-His-Gly-Cys-) were characteristic of the zygnematophycean PHYs known to date. There was no indication of a transmembrane region in Mougeotia phytochrome in particular, but a carboxyl-terminal 16-mer three-fold repeat in both, Mougeotia and Mesotaenium PHYs may represent a microtubule-binding domain. Unexpected for a non-angiosperm phytochrome, its expression was autoregulated in Mougeotia in a red/far-red reversible manner: under P_r conditions, phytochrome mRNA levels were tenfold higher than under P_fr conditions.     

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     

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)     

Light-Dependent Chloroplast Reorientation in Mougeotia and Mesotaenium: Riased by Pigment-Regulated Plasmalemma Anchorage Sites to Actin Filaments?

Conjugatophycean green algae, such as Mougeotia and Mesotaenium, are presumably the most ancient organisms to show phytochrome-mediated photomodulatory processes, i.e. chloroplast reorientational movements. Experiments have provided striking evidence for a dichroic mode of light absorption by the phytochrome molecules located at the periphery of the cylindrical cell; in addition, the transition moment of the chromophoric group of phytochrome has been shown to change by a fixed angle upon conversion of P_r to P_fr and vice versa. Consequently, a hypothesis has been put forward involving a tetrapolar phytochrome gradient at the plasmalemma. This presumed pigment pattern precisely controls chloroplast reorientation in the low-irradiance response. Intriguingly, a blue-light absorbing pigment is expressed in Mougeotia as well, which also mediates low-irradiance response via a presumed tetrapolar gradient, apparently independent of the phytochrome. Two hypotheses for the controlling mechanism of chloroplast reorientation have been put forward:

• a) Coupling of the influx of calcium through the plasmalemma to the tetrapolar gradient of the sensor pigment proper, resulting in a tetrapolar gradient of calcium in the cytoplasm. This is the “reorientation via calcium” hypothesis.
• b) Coupling of actin anchorage sites on the plasmalemma to the tetrapolar gradient of the sensor pigment proper, resulting in a tetrapolar gradient of actin anchorage sites. Cytoplasmic calcium, released from internal stores or taken up through the plasmalemma, triggers actomyosin interaction. This is the “reorientation via anchorage sites” hypothesis.

Consistent with the latter hypothesis, photoregulation by two steps seems to be indicated, (i) cytoplasmic initiation of actomyosin interaction, (ii) the graded formation of plasmalemma anchorage sites for actin filaments.     

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     

Partial purification and initial characterization of phytochrome from the mossAtrichum undulatum P. Beauv. grown in the light

The extraction and partial purification of phytochrome from light-grownAtrichum undulatum P. Beauv., a chlorophyllous moss, is described. Polyethyleneimine and salt fractionation followed by hydroxyapatite and Affi-gel-blue chromatography were used to separate phytochrome from chlorophyll, and to purify the pigment. All steps were performed in the presence of Triton X-100 which improved the yield by a factor of about three. The protein has a molecular weight some-what larger than that ofAvena phytochrome (124 kDa), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. It cross-reacts with a monoclonal antibody against phytochrome from etiolated corn (Zea) and a polyclonal antibody against phytochrome from etiolated oat (Avena), and its photoreversibility is similar to that of phytochrome from greenAvena.Abbreviations EDTA ethylenediaminetetraacetic acid - FMN flavinmononucleotide - PMSF phenylmethylsulfonylfluoride - P_r(P_fr) red(far-red)-absorbing form of phytochrome - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Mechanism of chloroplast movement inMougeotia

Summary The single, ribbon-shaped chloroplast in the filamentous green algaMougeotia performs orientational movements with respect to light. The chain of reaction involves phytochrome as the photoreceptor pigment to perceive the light signal differentiated by wavelength and direction, calcium probably to convert the light signal into a chemical message and actomyosin to respond to this message and to move the chloroplast accordingly.Precise reorientation of the chloroplast is proposed to be brought about by a dual function of phytochrome: regulation of the cellular level of calciumand regulation of membrane anchorage sites to actin.The Institute where ProfessorKamiya did early research together with ProfessorKüster.     

Cloning,Recombinant Expression and Characterization of Wild Type-105-Trp-Calmodulin of the Green Alga Mougeotia scalaris

The single calmodulin gene (CaM) of the green alga Mougeotia scalaris (Hassall) was cloned, sequenced and the CDNA inserted into the prokaryotic expression vector pGEX-2T. The recombinant calmodulin protein (CAM) was expressed as a fusion product together with glutathione S-transferase and isolated on glutathione sepharose. After cleavage and purification, the CaM was characterized by Ca²⁺-dependent shift in SDS-PAGE, by activation of cyclic 3′,5′nucleotide phosphodiesterase (PDE) and sensitivity to the inhibitors trifluoperazine and calmidazolium, with native Mougeotia CaM as control. Using Ca²⁺ buffers in the PDE test, affinity to Ca²⁺ of Mougeotia CaM was found to be diminished fivefold compared to maize or bovine brain CaMs. There was also a 20-fold increase of half maximal activation (K_act) in the PDE test for Mougeotia CaM relative to maize CaM, while the K_act of maize CaM to that of bovine brain CaM was almost the same. The derived amino acid sequences of CaM from Mougeotia and Zea mays revealed three major conservative amino acid exchanges, including unique 105-Trp (Mougeotia) → Leu (maize). In Mougeotia CaM the 105-Trp, including the neighbouring side chains of 92-Phe and 141-Phe, putatively form a hydrophobic ring interaction, as revealed by molecular modelling.     

Identification of calmodulin in the green alga Mougeotia and its possible function in chloroplast reorientational movement

A soluble protein was isolated from Mougeotia by chloropromazine-sepharose 4 B affinity chromatography. The protein matches the properties of calmodulin in terms of heat stability, Ca²⁺-dependent electrophoretic mobility in sodium-dodecyl-sulfate polyacrylamide gels, and its ability to activate cyclic nucleotide phosphodiesterase in a Ca²⁺-dependent manner. Phytochrome-mediated chloroplast reorientational movement in Mougeotia was inhibited by the calmodulin antagonist trifluoperazine, a hydrophobic compound, or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a hydrophilic compound; 50% inhibition (IC₅₀) of chloroplast movement is caused by 20–50 mol l^-1 trifluoperazine or 100 mol l^-1 W-7. The Ca²⁺-calmodulin may act as an intermediate in the chloroplast reorientational response in Mougeotia governed by phytochrome.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - SDS sodium dodecyl sulfate - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide     

Die Chloroplastendrehung beiMougeotia

Summary By means of partial illumination with small spots of monochromatic light, polarized obliquely to the cell axis, we could demonstrate that the phytochrome molecules inMougeotia are oriented in a spiral pattern around the cell surface. The lines of this spiral form an angle of about 45^o with the cell axis. The degree of exactness of this orientation has been discussed.

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Herrn Professor Dr.E. Heitz zum 70. Geburtstag.     

Cross-reactivity of monoclonal antibodies against phytochrome from Zea and Avena

The cross-reactivity of diverse monoclonal antibodies against phytochrome from Zea and Avena was tested by enzyme-linked immunosorbentassay (ELISA) and by immunoblotting. About 40 antibodies were selected by means of nondenatured phytochrome; all of them reacted with sodium dodecyl sulfate denatured homologous antigen on immunoblots. The epitopes for 14 antibodies (4 raised against Avena and 10 against Zea phytochrome) were localized in 6 regions of the phytochrome molecule by means of Western blot analysis of proteolytic fragments of known localization. Results of studies on the inhibition of antibody binding by other antibodies were largely compatible with these latter findings. Except in a few cases, inhibition occurred when antibodies were located on the same or a closely adjacent region. As demonstrated by 16 species, cross-reactivity with phytochromes from other Poaceae was high. Greater losses in cross-reactivity were observed only with antibodies recognizing an epitope in the vicinity of the carboxyl terminus of 118-kg · mol^-1 phytochrome. Cross-reactivity with phytochrome from dicotyledons was restricted to a few antibodies. However, phytochrome(s) from plants illuminated for 24 h or more could be detected. One of the antibodies that recognized phytochrome from dicotyledons was also found to recognize phytochrome or a protein of 120–125 kg·mol^-1 from several ferns, a liverwort and mosses. This antibody (Z-3B1), which was localized within a 23.5-kg·mol^-1 section of Avena phytochrome (Grimm et al., 1986, Z. Naturforsch. 41c, 993), seems to be the first antibody raised against phytochrome from a monocotyledon with such a wide range of reactivity. Even though epitopes were recognized on different phytochromes, the strength of antibody binding indicated that these epitopes are not necessarily wholly identical.Abbreviations ELISA enzyme-linked immunosorbent assay - McAb monoclonal antibody - PBS phosphate-buffered saline - Pfr (Pr) far-red-absorbing (red-absorbing) form of phytochrome - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

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 activation of k channels and chloroplast rotation in mougeotia: action spectra

The action spectra for K⁺ channel activation and chloroplast rotation are shown to be similar. Both phenomena exhibit activation at 660 nanometers, inhibition at 740 nanometers, and partial activation at 460 to 500 nanometers. This confirms that K⁺ channels in Mougeotia are regulated by phytochrome, and indicates that both phenomena share at least part of the same transduction pathway.     

A monoclonal antibody specific for the red-absorbing form of phytochrome

Characterisation of a new monoclonal antibody (mAb), designated LAS 41, directed against 124-kilodalton (kDa) etiolated-oat (Avena sativa L.) phytochrome, indicates that it recognises an epitope unique to the red-light-absorbing form, Pr. In a solid-phase enzyme-linked immunosorbent assay (ELISA), LAS 41 exhibits a seven- to eight-fold higher affinity for Pr than for the far-red-light-absorbing form of phytochrome, Pfr. In addition, in immunoprecipitation assays LAS 41 effectively precipitates 100% of phytochrome presented as Pr but only precipitates a maximum of 24.5% of phytochrome presented as Pfr. These values are indicative of binding exclusively to Pr. Peptide-mapping studies show that LAS 41 recognises and epitope located within a region 6–10 kDa from the aminoterminus of the phytochrome molecule. Since binding of LAS 41 to Pr induces alterations in the spectral properties of Pr, this indicates that at least part of the 4 kDa domain to which the antibody binds is essential for protein-chromophore interaction. Subsequent photoconversion of LAS 41-Pr complexes produces native Pfr spectra, with concomitant production of free antibody and antigen, as shown by a modified ELISA. The specificity of LAS 41 for Pr has facilitated the purification of Pfr which is free of contaminating Pr. This has enabled direct determination of the mole fraction of Pfr established by red light to be 0.874.Abbreviations ELISA enzyme-linked immunsorbent assay - kDa kilodalton - mAb monoclonal antibody - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - (A) difference in absorbance (A ₆₆₅ ^Pr –A ₇₃₀ ^Pr )-(A ₆₆₅ ^Pfr –A ₇₃₀ ^Pfr ) - Ar/Afr spectral change ratio (SCR) - max mole fraction of Pfr following saturating red light     

Phytochrome decay in seedlings under continuous incandescent light

Summary Under continuous high intensity incandescent light the decay of phytochrome in Amaranthus seedlings deviates from the predicted first order rate characteristic of the P _fr/P _total ratio maintained. This deviation takes the form of a slower decay than would be predicted and is only observed at high intensities. Experiments are presented to test the hypothesis that this reduced rate of decay is the result of a high level of phytochrome intermediates maintained under high intensity incandescent light. Accumulation of intermediates under these conditions has been demonstrated using a quasi-continuous measuring spectrophotometer. They are weakly absorbing and their concentration increases with light intensity. Although they form P _fr in darkness, it is proposed that they do not decay. The model predicts that in a sample cuvette, where a light intensity gradient exists, there is more probability of a phytochrome molecule being presnet as P _fr at the back of the cuvette: the region of lowest light intensity. Under conditions which favour phytochrome decay, a preferential loss of phytochrome should result at the back of the cuvette and an increasingly higher proportion of the remaining phytochrome will consequently be measured as intermediate as the experiment progresses. The results confirm the hypothesis and in addition, after 60 min incandescent light, demonstrate an accumulation of intermediates which form P _fr with a longer half-life that at the begining of the experiment. Pisum epicotyl hooks show no such intermediate accumulation or preferential decay at the back of the cuvette, which is in agreement with the observed first order phytochrome decay under high intensity incandescent light. A scheme is presented explaining the results on the basis of the decay process.Abbreviations FR far-red light - R red light - P phytochrome - P _fr far-red-absorbing form of P - P _r red-absorbing form of P 321st communication of this Laboratory.     

Native phytochrome: immunoblot analysis of relative molecular mass and in-vitro proteolytic degradation for several plant species

The relative molecular mass (M_r) of the native phytochrome monomer from etiolated Cucurbita pepo L., Pisum sativum L., Secale cereale L. and Zea mays L. seedlings has been determined using immunoblotting to visualize the chromoprotein in crude extracts subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single phytochrome band is observed for each plant species when the molecule is extracted under conditions previously demonstrated to inhibit the proteolysis of native Avena sativa L. phytochrome. A comparison among plant species indicates that the M_r of native phytochrome is variable: Zea mays=127000; Secale=Avena=124000; Pisum=121000; Cucurbita=120000. The in-vitro phototransformation difference spectrum for native phytochrome from each species is similar to that observed in vivo in each case and is indistinguishable from that described for native Avena phytochrome. The difference minima between the red- and far-red-absorbing forms of the pigment (Pr-Pfr) are all at 730 nm and the spectral change ratios (A_r/A_fr) are near unity. When incubated in crude extracts, phytochrome from all four species is susceptible to Pr-specific limited proteolysis in a manner qualitatively similar to that observed for Avena phytochrome, albeit with slower rates and with the production of different M_r degradation products. Further examination of the in-vitro proteolysis of Avena phytochrome by endogeneous proteases has identified several additional phytochrome degradation products and permitted construction of a peptide map of the molecule. The results indicate that both the 6000- and 4000-M_r polypeptide segments cleaved by Pr-specific proteolysis are located at the NH₂-terminus of the chromoprotein and are adjacent to a 64000-M_r polypeptide that contains the chromophore.Abbreviations and symbols A_minimum phototransformation difference spectrum (Pr-Pfr) minimum - Ig immunoglobulin - Mops 3-(N-morpholino)propanesulfonic acid - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate     

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.     

Gel filtration of particle-bound phytochrome

Pelletable phytochrome from hypocotyl hooks of Cucurbita pepo L. seedlings has been separated into two fractions by gel filtration on Sepharose CL-2B. One fraction with a K _av of 0.7 was detected only after red irradiation (in vivo or in vitro). This separated from a ribonucleoprotein fraction during gel filtration. The weak interaction with ribonucleo-protein which required magnesium (optimal at 10 mM) was overcome by high salt concentrations and prevented by ribonuclease treatment. The second phytochrome fraction was strongly associated with a high molecular weight material with a K _av of less than 0.1. Low levels of this complex were detected in extracts from dark grown tissue but were increased by red irradiation of excised hooks or crude extracts. The binding of phytochrome to the high molecular weight material did not require magnesium, was unaffected by ribonuclease treatment, and was much more resistant to high salt concentrations than was the phytochrome—ribonucleoprotein association. These results suggest that the association of phytochrome with this membrane—containing fraction is not electrostatic.The separation by agarose-gel filtration offers a useful technique for the preparation of membraneassociated phytochrome for physiological studies.Abbreviations EDTA ethylenediaminetetraacetic acid - P _r and P_fr phytochrome in the red and far-red absorbing forms - RNase ribonuclease - RNP ribonucleoprotein     

Phytochrome in seeds of Amaranthus caudatus

Summary Dry seeds of Amaranthus caudatus show little or no photoreversible absorption changes, attributable to phytochrome. During imbibition phytochrome appears in two phases, one immediately after sowing and the second after about 8 hr. Experiments at different temperatures and under continuous illumination with red, far-red and blue light suggest that there are two pools of phytochrome. The first phase in the appearance of phytochrome could be due to the change in optical properties of the sample on hydration or to rehydration of inactive phytochrome, or both. The second phase probably represents phytochrome synthesis. It is absent at 0° and precedes the water uptake associated with germination by some 10 hr. This second pool of phytochrome does not accumulate in red and blue illuminated seeds indicating that the rate of P _fr decay is more rapid than the rate of phytochrome synthesis. The difference spectra of phytochrome in both 2 hr imbibed seeds and 72 hr old seedlings show peaks of absorption at 663 and 735 nm. The presence of P _fr in dark imbibed seeds and the process of inverse reversion of P _r to P _fr in darkness have been demonstrated. The results are discussed in relation to previous hypotheses for the mechanism of photocontrol of Amaranthus seed germination.     

Stoichiometric and growth responses of a freshwater filamentous green alga to varying nutrient supplies: slow and steady wins the race

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