Red Light-Induced Appearance of Phosphotyrosine-like Epitopes on Nuclear Proteins from Pea (Pisum sativum L.) Plumules

Abstract— As assayed by western blot analysis, red light induces the appearance of epitopes recognized by anti-phosphotyro-sine antibodies in several pea nuclear proteins. The im-munostaining is blocked by preadsorbing the antibodies with phosphotyrosine but not by preadsorbing them with phosphoserine or phosphothreonine. This light response is observed whether the red light irradiation is given to pea plumules or nuclei isolated from the plumules. The red-light-induced response seen in plumules is reversible by a subsequent far-red-light irradiation, indicating that the likely photoreceptor for this response may be phy-tochrome. By immunoblot analysis pea phytochrome A, but not phytochrome B, can be detected in proteins extracted from pea nuclear chromatin-matrix preparations. Phytochrome A and the protein bands immuno-stained by anti-phosphotyrosine antibodies can be solu-bilized from unirradiated pea chromatin by 0.3 M NaCl, but irradiating this preparation with red light does not induce the appearance of phosphotyrosine-like epitopes in any nuclear proteins. These results suggest that the association of phytochrome with purified pea nuclei is such that its conversion to the far-red light-absorbing form can induce a post-translational epitope change in nuclear proteins in vivo .     

PHYTOCHROME CONTROL OF ITS OWN SYNTHESIS IN Sorghum vulgare AND Avena sativa

The accumulation of phytochrome in the dark was measured for Avena sativa seedlings after a white light pretreatment and for Sorghum vulgare seedlings after continuous red or far-red light treatments, using the herbicide Norflurazon to prevent greening under continuous irradiation. In both cases the accumulation of phytochrome depends on the state of the phytochrome at the light-dark transition: high P_fr levels (red light pulse) led to a slower rate of phytochrome accumulation than lower P_fr levels (long wavelength far-red (RG 9) light pulse). Poly-(A⁺)-RNA was isolated fromA. sativa seedlings grown for 48 h in darkness + 24 h WL + light pulse (5 min) (red, RG 9 light, red followed by RG 9 light or RG 9 followed by red light pulse) + 19 h darkness. The poly-(A⁺)-RNA was translated in a rabbit reticulocyte lysate system and the translation products were immunoprecipitated by specific anti-phytochrome antibodies. It was demonstrated that the activity of mRNA coding for phytochrome was under phytochrome control.     

THE PHOTORECEPTORS INVOLVED IN ANTHOCYANIN SYNTHESIS

Abstract— Experiments with irradiation sequences where red precedes far-red lead to the conclusion that, in turnip, phytochrome is the only pigment mediating anthocyanin synthesis in red and far-red. Results from experiments where far-red precedes red, however, suggest that more than one reaction is involved. A possible interpretation is that the 'high-energy' reaction in far-red and the low energy red/far-red reversible reaction are mediated by two different forms of phytochrome.
The 'high-energy' reaction in blue light does not appear to depend on phytochrome.     

APPARENT INCREASE IN PHOTODETECTABLE PHYTOCHROME IN THE PRESENCE OF CALCIUM CARBONATE*

Abstract— –Using squash seedling extracts ( Cucurbita pepo L.), we describe an apparent increase in photodetectable phytochrome content that is depended upon the use of either CaCO₃ or starch as a scattering agent and is also a function of preirradiation with red light of either intact tissue or crude extracts prior to sample preparation for spectrophotometric assay. This apparent increase in photodetectable phytochrome content requires about 1 h for full expression after addition of the scattering agent. If irradiations are given in vitro , the increase is partially reversed by far red light when using squash extracts, and is fully reversed with Aoena and Zea extracts. For squash extracts the magnitude of this increase. which is typically between 20 and 40%. is quantitatively correlated with both the amount of Pfr produced by a brief red irradiation and with the proportion of the total phytochrome pool that is pelletable upon centrifugation at 20,000 g . The correlation with pelletable phytochrome does not hold for Auena and Zea , at least when irradiations are gwen in uitro . The increase in photodetectable phytochrome may result from changed phytochrome extinction, changed phototransformation quantum yields, and/or specific trapping of phytochrome by the scattering agent as it settles. An important consequence of these data is that they indicate a need for caution when using a scattering agent during spectrophotometric assay of phytochrome.     

PHOTOREVERSIBLE Ca2+-DEPENDENT AGGREGATION OF PURIFIED PHYTOCHROME FROM ETIOLATED PEA AND RYE SEEDLINGS

The aggregation of phytochrome purified from etiolated pea ( Pisum satirum cv. Alaska) and rye ( Secale cereale cv. Cougar) tissues was investigated by centrifugation and turbidimetry. Purified pea phytochrome (A₆₆₉/A₂₈₀= 0.88), if irradiated with red light, became precipitable in the presence of CaCl₂. The precipitation upon red-light irradiation was optimal at a Ca^2- or Mg²⁺ concentration of 10–20 m M , was greater at increased phytochrome concentration or lower pH values, and was inhibited by 0.1 M KG. The precipitated phytochrome slowly became soluble after far-red light exposure.
Turbidity of pea phytochrome solutions after red-light irradiation also increased rapidly in the presence of either Ca²⁺ or Mg²⁺. Far-red light exposure after the red light cancelled the turbidity increase. Rye phytochrome showed less turbidity increase than pea phytochrome and occurred only in the presence of Ca²⁺. Partially degraded pea phytochrome produced by endogenous proteases in the extract did not show the turbidity increase. Undegraded pea phytochrome also associated with microsomal fractions under conditions similar to those described above, but the partially degraded phytochrome did not.     

INTERACTION BETWEEN PHYTOCHROME AND THE BLUE LIGHT PHOTORECEPTOR SYSTEM IN Mougeotia*

Abstract— Face-to-profile chloroplast movement in Mougeotia was induced by sequences of strong blue and red short irradiations. This type of response occured only when blue light was applied prior to or simultaneously with red light, and far-red irradiation was necessary after the sequence to cancel the remaining gradient of the far-red absorbing form of phytochrome P_fr. The dependence of the response magnitude on blue and red light sequences was studied for a wide range of light durations and dark intervals. The relationship between the response and the dark interval points to the lack of direct coupling between phytochrome and blue-absorbing “cryptochrome”. It was postulated that a photoproduct having a life-time of2–3 min is formed by the blue-light-mediated reaction. This photoproduct interacts with phytochrome during its transformation or with its final P_fr form.     

LIGHT INDUCED FLUORESCENCE SPECTRAL CHANGES IN NATIVE PHYTOCHROME FROM Secale cereale L. AT LIQUID NITROGEN TEMPERATURE

Abstract— Fluorescence spectra of native rye phytochrome were determined under different light conditions at liquid nitrogen temperature. Fluorescence spectrum of the red-light-absorbing form (Pr) had a major peak at about 685 nm (14 600 cm⁻¹) and a broad sub-peak at about 515 nm (19 400 cm⁻¹). The peak height at 685 nm was reduced by irradiation with monochromatic light of 640 nm, and a new peak became obvious at about 702 nm (14250 cm⁻¹). This spectral change was almost completely reversed by subsequent irradiation with 700-nm light. Fluorescence spectrum of the photoequilibrium mixture of Pr and far-red-light absorbing form under continuous red light showed a sharp peak at about 685 nm having a peak height ca. 12% of Pr, and a broad sub-peak at about 508 nm (19 700 cm⁻¹). Light of 730 nm did not reduce the peak height at about 685 nm but induced a new shoulder at about 699 nm (14300 cm⁻¹). Monochromatic light of 640 and 700 nm given following the light of 730 nm could not reverse the spectral change at 699 nm induced by the irradiation with 730-nm light. Fluorescence spectrum of Pr in partially degraded phytochrome was similar to that in native phytochrome but the peak position in the red region was shifted by about 5 nm (100 cm⁻¹) to the blue.     

Identification of a light-regulated protein kinase activity from seedlings of Arabidopsis thaliana

Protein kinase transduction pathways are thought to be involved in light signaling in plants, but other than the photoreceptors, no protein kinase activity has been shown to be light-regulated in vivo. Using an in-gel protein kinase assay technique with histone H III SS as an exogenous substrate, we identified a light-regulated protein kinase activity with an apparent molecular weight ca 50 kDa. The kinase activity increased transiently after irradiation of dark-grown seedlings with continuous far red light (FR) and blue light (B) and decreased after irradiation with red light (R). The maximal activation was achieved after 30 min to 1 h with FR or B. After irradiation times longer than 2 h, the kinase activity decreased to below the sensitivity level of the assay. In Arabidopsis mutants lacking either the photoreceptors phytochrome A, phytochrome B or the blue-light receptor cryptochrome 1, kinase activity was undetectable, whereas in the photomorphogenic mutants cop1 and det1 the kinase activity was also observed in the absence of light signals, though still stimulated by B and FR. Interestingly, the R inhibition of the kinase activity was lost in the mutant hy5. Pretreatment with cycloheximide blocked the kinase activity.     

PHYTOCHROME MACRO-DISTRIBUTION, LOCAL PHOTOCONVERSION AND INTERNAL PHOTON FLUENCE RATE FOR Cucurbita pepo L. COTYLEDONS

Abstract— Using 7-day-oId cotyledons of Cucurbita pepo L., local phytochrome photoconversions could be measured for blue, red and far-red light. For this purpose, after nonsaturating irradiation, cotyledons were sliced into discs 0.3 to 0.5 mm thick and signals measured. This method also yielded the internal phytochrome distribution of the cotyledons with maximal concentration near the adaxial surface, dropping to about 50% in the center and reaching again about 90% at the abaxial surface. Local phytochrome conversion rates were used to calculate internal fluence rates across the cotyledons. Relative internal fluence rates were also derived from measured reflectances and transmittance according to the Kubelka-Munk theory. The general shape of the internal fluence distribution calculated on the basis of these two methods coincided well. It was observed that the internal local photoconversion is proportional to the penetration depth over a wide range of incident fluences and for all wavelengths tested, showing in addition that reciprocity holds. A method to calculate internal fluence rates by a simplified procedure assuming either linear or exponential functions is described.     

Spectroscopic detection of a phytochrome-like photoreceptor in the myxomycete Physarum polycephalum and the kinetic mechanism for the photocontrol of sporulation by Pfr.

Sporulation of the true slime mold Physarum polycephalum (Myxomycetales) can be triggered by the far-red/red reversible Physarum phytochrome. Physarum plasmodia were analyzed with a purpose-built dual-wavelength photometer that is designed for phytochrome measurements. A photoreversible absorbance change at 670 nm was monitored after actinic red (R) and far-red (FR) irradiation of starved plasmodia, confirming the occurrence of a phytochrome-like photoreceptor in Physarum spectroscopically. These signals were not found in growing plasmodia, suggesting the Physarum phytochrome to be synthesized during starvation, which makes the cells competent for the photoinduction of sporulation. The photoconversion rates by R and FR light were similar in the phytochromes of Physarum and etiolated oat shoots. In dark-grown Physarum plasmodia that had not been preexposed to any light only R induced a detectable absorbance change while FR did not. This indicates that most (at least 90%) of the photoreversible pigment occurs in the red-absorbing form. Since the effectiveness of FR in triggering sporulation was enhanced by preirradiation with R, it is concluded that at least part of the Pr can be photoconverted to the active Pfr photoreceptor species. We propose a kinetic mechanism for the photocontrol of sporulation by photoconversion of Pfr, which may also hold for the high-irradiance response to FR in Arabidopsis and Cuscuta.     

PHYTOCHROME INDUCTION OF PHOTOREACTIVATING ENZYME IN Phaseolus vulgaris L. SEEDLINGS*

Abstract— Photoreactivating enzyme (PRE) activity was measured in hypocotyls of Phaseolus vulgaris L. seedlings using a radioimmunoassay for thymine dimers. In dark-grown seedlings a five-fold increase in PRE activity was observed after 6 h of irradiation with blue or far-red light. Short time irradiations with red light were also effective. Reversibility of this red-light-effect by a subsequent short term irradiation with far-red light and also the high effectiveness of continuous far-red light indicate that PRE activity is under phytochrome control. This observation points to PRE induction via gene activation.     

Phytochrome-regulated Starch Degradation in Germinating Turions of Spirodela polyrhiza

Abstract— Turions of the duckweed Spirodela polyrhiza contain about 70% starch on the dry weight basis. The rate of starch degradation in nondormant turions was highest in continuous red light (cR) followed by continuous blue light (cB), whereas continuous far red light (cFR) is almost ineffective. Continuous R could be substituted by repeated R pulses; this effect was not photoreversible by FR pulses applied after hourly R pulses. This suggested that R-dependent high irradiance response is the mode of phytochrome action in mediating starch degradation. Comparing the mode of phytochrome action with that of phytochrome mutants of other plants it can be assumed that phytochrome B is the photoreceptor involved. Application of the translation inhibitor cycloheximide immediately stopped the phytochrome-dependent starch degradation demonstrating the involvement of de novo synthesis of proteins.     

EVIDENCE FOR PHYTOCHROME CONTROLLED ENDOMITOSIS AND CELL ELONGATION IN PISUM SATIVUM EPICOTYLS

Elongation and endomitosis were studied in the epicotyl cortex cells of germinating seeds of Pisum sativum cv. Rondo. One min of red light per 24 h is sufficient to fully inhibit endomitosis. Terminal far-red irradiation can reverse the red effect to the level established by far-red light alone. This justifies the conclusion that phytochrome is involved in the regulation of endomitotic DNA replication. Since far-red light alone inhibits endomitosis to about 50%, we conclude that very low levels of Pfr are required to influence the endomitotic cycle.     

PHYTOCHROME IN ETIOPLASTS IN RELATION TO CHLOROPHYLL ACCUMULATION*

Abstract— De-etiolation of maize seedlings reduces their sensitivity for red light potentiation of rapid chlorophyll accumulation in white light. An earlier proposal (Raven and Spruit, 1973) attributes this to migration of the far-red absorbing form of phytochrome (P_fr) to receptors essential for chlorophyll synthesis, thereby increasing the local P_fr/total phytochrome (P_tot)ratio. We have studied etioplasts as possible loci for such P_(r receptors. The level of spectrophotometric phytochrome in purified etioplasts isolated from red preirradiated maize seedlings was higher than that of dark grown plants. The difference was marginally significant, however. We argue that migration of a fraction of cytoplasmic P_fr to the etioplasts, too small to be spectrophotometically demonstrable, could still meet the requirements of the model. Dark destruction of bulk spectrophotometric P_fr following saturating red irradiation of seedlings is not paralleled by a decrease of etioplast phytochrome. the latter remaining essentially constant over long periods. On the other hand, the potentiating effect of red light in intact seedlings is still partially reversible by far red light even after 24 h of darkness when destruction of bulk P_fr is complete. Since this demonstrates persistent presence of P_fr active in potentiation, we propose that at least part of this P_fr is associated with the etioplasts.     

PHOTOREVERSIBLE CHANGES IN pH OF PEA PHYTOCHROME SOLUTIONS

Abstract— Photoinduced pH changes in unbuffered solutions of undegraded pea phytochrome were studied at 10_oC by using a glass electrode. Red light irradiation caused alkalinization of the solutions in the pH range 5.2–xs7.5 and acidification in pH 7.5–8.9. The pH changes were fully reversed by a subsequent irradiation with far-red light. The red and far-red light effects were repeatedly reversible. The solution of tryptic peptide of phytochrome (mol. wt 60000) showed similar photoreversible pH changes.     

PHYTOCHROME MEDIATED CAROTENOID SYNTHESIS IN THE FUNGUS VERTICILLIUM AGARZCZNUM

Abstract— Ten minutes of red irradiation (R) increased carotenogenesis in Verticillium agoricinum and this effect was reversed by 10min of far-red (FR) irradiation indicating that phytochrome is involved. A far-red minus red difference spectrum of a crude extract shows a peak at 670 nm and a dip at 750 nm wavelength, values slightly larger than higher plant phytochrome. indicating the presence of phytochrome.     

PHYTOCHROME CONTROL OF THE DEVELOPMENT OF PHOTOPHOSPHORYLATION

In the cotyledons of the mustard (Sinapis ah L.) seedling the development of the capacity for photophosphorylation is strongly influenced by pretreatment of the seedling with red light pulses. The red light acts through phytochrome. After a red light pretreatment the capacity for photophosphorylation increases linearly with the chlorophyll content, at least up to 30 min after the onset of continuous white light. It is proposed that the reaction chain required for photophosphorylation is completed under the influence of phytochrome even in the absence of chlorophyll. As soon as chlorophyll becomes available photophosphorylation functions instantaneously. Without a red light pretreatment there is a lag of more than 15 min before photophosphorylation becomes detectable after the onset of continuous white light even though chlorophyll a is available. Although phytochrome strongly influences the rate of chlorophyll accumulation as well it is improbable that the control by phytochrome of development of photophosphorylation and of chlorophyll accumulation are causally connected.     

THE CONTROL BY PHYTOCHROME OF NITRATE REDUCTASE IN THE CURD OF LIGHT-GROWN CAULIFLOWER

Abstract— The activity of nitrate reductase from the curd of light-grown cauliflower ( Brassica oleracea (L) var botrytis (DC) 'St. Hilary') is modulated by nitrate and by light. Using broad-band sources of equal photosynthetically active radiation but with different proportions of red and far-red light, a linear relationship between nitrate reductase activity and ψ(Estimated phytochrome photoequilibrium) was obtained. This relationship, apparent after 8 h incubation, was maintained and little altered after 48 h incubation. The linearity was apparent between ψE 0.26 and ψE 0.69; ψE 0.26 being no more effective than a dark control. Far-red reversibility confirmed the involvement of phytochrome. Brief pulses of red light were also used to establish a range of phytochrome photoequilibria within the tissue. Again a linear relationship between ψ and nitrate reductase activity was obtained with a threshold for the response at ψ 0.3. With both monochromatic and broad-band sources it was seen that neither photon fluence rate nor duration of exposure affected the final activity of the enzyme and that phytochrome was acting solely through ψ (or [Pfr] since phytochrome is stable in this tissue) to bring about these responses.     

CONTROL BY PHYTOCHROME OF EXTENSION GROWTH and POLAROTROPISM IN CHLORONEMATA OF Funaria hygrometrica

Abstract— The photocontrol of extension growth and polarotropism has been investigated in chloronemata of the moss Funaria hygrometrica Hedw. When grown on a 12/12 h light/dark cycle, chloronemata show light-stimulated diurnal variations in elongation rate with no evidence of a circadian rhythm. Regulation of elongation by a low energy, photoreversible phytochrome mechanism was demonstrated by brief red (R) and far red (FR) irradiations given either at the end of the day (EOD) or 6 h later as a night break (NB). Night break irradiation with polarised R caused polarotropic reorientations of chloronemal growth also through a FR-reversible, low energy phytochrome mechanism. Such transformations of P_r to P_fr were accompanied by a 90° shift in the orientation of the phytochrome chromophore. Microbeam irradiation indicated that the phytochrome chromophores involved in both responses were predominantly, if not entirely, located in the tip of the apical chloronemal cell.     

RED/FAR-RED PHOTOREVERSIBILITY: NOT AN APPROPRIATE PHYTOCHROME ASSAY FOR RED-LIGHT PREIRRADIATED CORN COLEOPTILES

Abstract— Based on measurements with a single beam spectrophotometer, it has been found that subsequent red/far red irradiation cycles, which are usually given to monitor phytochrome content by dual wavelength spectroscopy, induce chlorophyll-related absorption changes in maize coleoptiles. Therefore, the difference signal, usually measured between 730 and 800 nm or 660 and 730 nm after saturating red and far red irradiations, does not represent solely the phytochrome content of preirradiated samples.