Red light-induced accumulation of ubiquitin-phytochrome conjugates in both monocots and dicots

Phytochrome is rapidly degraded in vivo after photoconversion from the stable red-absorbing (Pr) form to the far red-absorbing (Pfr) form. Previously, we have shown in etiolated oat seedlings that ubiquitin-phytochrome conjugates (Ub-P) appear after Pfr formation suggesting that oat phytochrome is rapidly degraded by a ubiquitin-dependent proteolytic pathway. Here, we extend this observation to etiolated tissue from other monocotyledonous (corn [Zea mays. (L.)] and rye [Secale cereale (L.)] and dicotyledonous species (pea [Pisum sativum (L,)] and zucchini squash [Cucurbita pepo (L.)]). Following Pfr formation by red light, all four species synthesized a heterogeneous series of Ub-P that appeared and disappeared concomitant with the degradation of the chromoprotein. When Pfr was photoconverted back to Pr by a far-red light pulse, degradation of phytochrome ceased and the levels of Ub-P concomitantly dropped. In pea and zucchini squash, loss of Ub-P after photoconversion of Pfr back to Pr was rapid, occurring with a half-life of approximately 5 to 10 minutes. These data indicate that the accumulation of Ub-P after Pfr formation is a general phenomenon in etiolated seedlings of higher plants and further support the hypothesis that plants degrade Pfr via Ub-P intermediates.     

Intracellular localisation of phytochrome and ubiquitin in red-light-irradiated oat coleoptiles by electron microscopy

The intracellular localisation of phytochrome and ubiquitin in irradiated oat coleoptiles was analysed by electron microscopy. We applied indirect immunolabeling with polyclonal antibodies against phytochrome from etiolated oat seedlings or polyclonal antibodies against ubiquitin from rabbit reticulocytes, together with a goldcoupled second antibody, on serial ultrathin sections of resin-embedded material. Immediately after a 5-min pulse of red light-converting phytochrome from the red-absorbing (Pr) to the far-redabsorbing (Pfr) form-the label for phytochrome was found to be sequestered in electron-dense areas. For up to 2 h after irradiation, the size of these areas increased with increasing dark periods. The ubiquitin label was found in the same electrondense areas only after a dark period of 30 min. A 5 min pulse of far-red light, which reverts Pfr to Pr, given immediately after the red light did not cause the electron-dense structures to disappear; moreover, they contained the phytochrome label immediately after the far-red pulse. In contrast, after the reverting far-red light pulse, ubiquitin could only be visualised in the electron-dense areas after prolonged dark periods (i.e. 60 min). The relevance of these data to light-induced phytochrome pelletability and to the destruction of both Pr and Pfr is discussed.Abbreviations FR far-red light; Pfr - Pr far-red-absorbing and red-absorbing forms of phytochrome, respectively - R red light     

Integral association of phytochrome with a membranous fraction fromAvena shoots: in vivo characterization and physiological significance

Phytochrome in the far-red light absorbing form (Pfr) was observed to disappear in vivo more rapidly from the non-cation-requiring pelletable phytochrome population than from the supernantant phytochrome population of oat seedlings given an increasing dark incubation after red irradiation. The amount of pelletable phytochrome in the red light absorbing form (Pr) remained relatively stable while supernatant Pr was lost. These observations indicated that supernant Pfr was subject to loss during the incubation, while pelletable Pfr was subject to both dark reversion and loss.During the incubation, the ability of far-red irradiation to reverse the red-induced increase in phytochrome pelletability was lost, with kinetics similar to those of the loss of pelletable Pfr.Far-red reversibility of the red-induced increase in coleoptile elongation correlated with the change intotal Pfr in both supernatant and pelletable phytochrome populations, but with the change in the ratio of Pfr to total phytochrome only in the pelletable phytochrome population.The possible significance of these results is discussed with reference to the action of phytochrome in the photocontrol of physiological growth responses.Abbreviations Pfr phytochrome in the far-red light absorbing form - Pr phytochrome in the red absorbing form - Ptot total phytochrome     

Intracellular localisation of phytochrome in oat coleoptiles by electron microscopy

The intracellular localisation of phytochrome in oat (Avena sativa L. cv. Garry Oat) coleoptiles was analysed by electron microscopy. Serial ultrathin sections of resin-embedded material were indirectly immunolabeled with polyclonal antibodies against phytochrome together with a gold-coupled second antibody. The limits of detectability of sequestered areas of phytochrome (SAPs) were analysed as a function of light pretreatments and amounts of the far-red absorbing form of phytochrome (Pfr) established. In 5-d-old dark-grownAvena coleoptiles SAPs were not detectable if less than 13 units of Pfr — compared with 100 units total phytochrome of 5-d-old dark-grown seedlings — were established by a red light pulse. In other sets of experiments, seedlings were preirradiated either with a non-saturating red light pulse to allow destruction to occur or with a saturating red followed by a far-red light pulse to induce first SAP formation and then its disaggregation. These preirradiations resulted in an increase of the limit of detectability of SAP formation after a second red light pulse to 38–41 and 19–23 units Pfr, respectively. We conclude that with respect to Pfr-induced SAP formation an adaptation process exists and that our data indicate that SAP formation is not a simple self-aggregation of newly formed Pfr.Abbreviations FR far-red light - Pfr, Pr far-red-absorbing and red-absorbing forms of phytochrome, respectively - Plot total phytochrome (Pfr + Pr) - R red light - SAP sequestered areas of phytochrome This work was supported by Deutsche Forschungsgemeinschaft (SFB 206). The competent technical assistance of Karin Fischer is gratefully acknowledged.     

Visualization of a rapid,red/far-red light-dependent reaction by centrifuge microscopy

Summary Using a centrifuge microscope with stroboscopic illumination, we examined the effects of light irradiation on the passive movement of chloroplasts in dark-adapted mesophyll cells ofVallisneria gigantea. While irradiation with red light accelerates the passive gliding of chloroplasts produced by centrifugal force, irradiation with far-red light negates this effect. Irradiation with blue light does not accelerate the passive gliding, while red light is completely effective even in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosynthesis. An apparently active movement of chloroplasts can be induced by irradiation with red or blue light only in the presence of the far-red light-absorbing form of phytochrome. The significance of the reaction in the light with respect to the regulation of cytoplasmic streaming is discussed.Abbreviations APW artificial pond water - CMS centrifuge microscope of the stroboscopic type - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Pfr phytochrome, far-red light-absorbing form - Pr phytochrome, red light-absorbing form     

Dynamic properties of endogenous phytochrome A in Arabidopsis seedlings.

The dynamic behavior of phytochrome A (phyA) in seedlings of the model plant Arabidopsis was examined by in vivo spectroscopy and by western and northern blotting. Rapid accumulation of phyA was observed, reaching a steady state after 3 d. Both red and far-red light initiated a rapid destruction of the far-red-light-absorbing form of phytochrome (Pfr); the apparent half-life was only 4-fold longer in far-red than in red light. Furthermore, the Pfr-induced destruction of the red-light-absorbing form of phytochrome (Pr) of phyA occurred in darkness with a rate identical to that of Pfr destruction. A 2-fold decrease in mRNA abundance was observed after irradiation, irrespective of the applied light quality. However, reaccumulation occurred rapidly after far-red but slowly after red irradiation, indicating different modes of regulation of phytochrome expression after light-dark transitions depending on the light quality of the preceding irradiation. The wavelength dependency of the destruction rates was distinct from that of mustard, a close relative of Arabidopsis, and was explained on the basis of Pfr-induced Pr destruction and a simple kinetic two-step model. No dark reversion was detectable in the destruction kinetics after a red pulse. From these data we conclude that Arabidopsis phyA differs significantly in several aspects from other dicot phytochromes.     

Phytochrome-specific type 5 phosphatase controls light signal flux by enhancing phytochrome stability and affinity for a signal transducer

Environmental light information such as quality, intensity, and duration in red (approximately 660 nm) and far-red (approximately 730 nm) wavelengths is perceived by phytochrome photoreceptors in plants, critically influencing almost all developmental strategies from germination to flowering. Phytochromes interconvert between red light-absorbing Pr and biologically functional far-red light-absorbing Pfr forms. To ensure optimal photoresponses in plants, the flux of light signal from Pfr-phytochromes should be tightly controlled. Phytochromes are phosphorylated at specific serine residues. We found that a type 5 protein phosphatase (PAPP5) specifically dephosphorylates biologically active Pfr-phytochromes and enhances phytochrome-mediated photoresponses. Depending on the specific serine residues dephosphorylated by PAPP5, phytochrome stability and affinity for a downstream signal transducer, NDPK2, were enhanced. Thus, phytochrome photoreceptors have developed an elaborate biochemical tuning mechanism for modulating the flux of light signal, employing variable phosphorylation states controlled by phosphorylation and PAPP5-mediated dephosphorylation as a mean to control phytochrome stability and affinity for downstream transducers.     

An unusual effect of the far-red absorbing form of phytochrome: Photoinhibition of seed germination inBromus sterilis L.

Seeds ofBromus sterilis L. germinated between 80–100% in darkness at 15° C but were inhibited by exposure to white or red light for 8 h per day. Exposure to far-red light resulted in germination similar to, or less than, that of seeds maintained in darkness. Germination is not permanently inhibited by light as seeds attain maximal germination when transferred back to darkness. Germination can be markedly delayed by exposure to a single pulse of red light following 4 h inhibition in darkness. The effect of the red light can be reversed by a single pulse of far-red light indicating that the photoreversible pigment phytochrome is involved in the response. The response ofB. sterilis seeds to light appears to be unique; the far-red-absorbing form of phytochrome (Pfr) actually inhibiting germination.Abbreviations Pr red absorbing form of phytochrome - Pfr far-red absorbing form of phytochrome     

The kinetics of type 1 phytochrome in green,light-grown wheat (Triticum aestivum L.)

The kinetics of type 1 phytochrome were investigated in green, light-grown wheat. Phytochrome was measured by a quantitative sandwich enzyme-linked immunosorbent assay using monoclonal antibodies. The assay was capable of detecting down to 150 pg of phytochrome. In red light, rapid first-order destruction of the far-red-light-absorbing form of phytochrome (Pfr) with a half-life of 15 min was observed. Following white light terminated by red, phytochrome synthesis was delayed in darkness by about 15 h compared to plants given a terminal far-red treatment. Synthesis of the red-light-absorbing form of phytochrome (Pr) was zero-order in these experiments. Phytochrome synthesis in far-red light was approximately equal to synthesis in darkness in wheat although net destruction occurred in light-grown Avena sativa tissues in continuous far-red light, as has been reported for other monocotyledons. In wheat, destruction of Pfr apparently did not occur below a certain threshold level of Pfr or Pfr/total phytochrome. These results are consistent with an involvement of type 1 phytochrome in the photoperiodic control of flowering in wheat and other long-day plants.Abbreviations ELISA enzyme-linked immunosorbent assay - FR far-red light - HIR high-irradiance response - Pfr farred-light-absorbing form of phytochrome - Pr red-light-absorbing form of phytochrome - Ptot total phytochrome (Pr + Pfr) - R red light The authors wish to thank Prof. Daphne Vince-Prue (University of Reading) for many helpful discussions regarding this work. Hugh Carr-Smith was supported by a Science and Engineering Research Council studentship and Chris Plumpton by an Agricultural and Food Research Council (AFRC) studentship. B. Thomas and G. Butcher were supported by the AFRC.     

N-terminal domain of Avena phytochrome: interactions with sodium dodecyl sulfate micelles and N-terminal chain truncated phytochrome.

Phytochrome is the ubiquitous red light photoreceptor present in plants. Properties of the 6-kDa end terminal region of phytochrome A (PHYA from etiolated Avena) have been investigated by the use of synthetic polypeptide fragments corresponding to that region. This region of the phytochrome A protein has been viewed as a possible functional site due to the large differences in the sequence's conformation and exposure between the Pr (red light-absorbing form) and Pfr (far-red light-absorbing, gene-regulating form) species of phytochrome A. Hydrophobic moment calculations reveal amphiphilic helical potential in this section of the protein, consistent with the folding of the N-terminal region onto a hydrophobic chromophore/chromophore pocket. A large N-terminal synthetic peptide also demonstrated helical folding in the presence of SDS micelles. This experimental evidence indicates that the N-terminal alpha-helical folding upon conversion of the regulatorily inactive Pr to the active Pfr form of phytochrome A is likely driven at least in part by amphiphilic helix stabilization. Further, the large synthetic peptide was spectrally demonstrated to interact with phytochrome A lacking the N-terminal region. The formation of this nativelike complex may provide us with a tool for both biophysical and physiological studies on the mechanism of phytochrome A signal transduction.     

Both subunits of the dimeric plant photoreceptor phytochrome require chromophore for stability of the far-red light-absorbing form

The dimeric plant photoreceptor phytochrome is converted from its inactive red light-absorbing form (Pr) into the active far-red light-absorbing form (Pfr) upon light absorption. Dynamics of Pfr generation and of thermal Pfr-to-Pr conversion are of fundamental importance for inducing adequate responses to light signals. Here, we analyzed the role of subunit interactions on spectroscopic properties of dimeric phytochrome A. Using a coexpression system and affinity chromatography, we prepared mixed phytochrome dimers that can incorporate the essential chromophore only in one subunit. We demonstrate that such mixed dimers have unaltered difference spectra. In contrast, dark reversion differed greatly between Pfr-Pfr homodimers and Pfr-Pr heterodimers, the former being about 100-fold more stable. Temperature dependence of reaction rates revealed an additional stabilization of about 4 kcal/mol in homodimers. Consequences of these findings are discussed in relation to the biological function of, and functional diversification between, phytochrome family members.     

Characterization of two thermostable cyanobacterial phytochromes reveals global movements in the chromophore-binding domain during photoconversion

Photointerconversion between the red light-absorbing (Pr) form and the far-red light-absorbing (Pfr) form is the central feature that allows members of the phytochrome (Phy) superfamily to act as reversible switches in light perception. Whereas the chromophore structure and surrounding binding pocket of Pr have been described, those for Pfr have remained enigmatic for various technical reasons. Here we describe a novel pair of Phys from two thermophilic cyanobacteria, Synechococcus sp. OS-A and OS-B', that overcome several of these limitations. Like other cyanobacterial Phys, SyA-Cph1 and SyB-Cph1 covalently bind the bilin phycocyanobilin via their cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) domains and then assume the photointerconvertible Pr and Pfr states with absorption maxima at 630 and 704 nm, respectively. However, they are naturally missing the N-terminal Per/Arndt/Sim domain common to others in the Phy superfamily. Importantly, truncations containing only the GAF domain are monomeric, photochromic, and remarkably thermostable. Resonance Raman and NMR spectroscopy show that all four pyrrole ring nitrogens of phycocyanobilin are protonated both as Pr and following red light irradiation, indicating that the GAF domain by itself can complete the Pr to Pfr photocycle. (1)H-(15)N two-dimensional NMR spectra of isotopically labeled preparations of the SyB-Cph1 GAF domain revealed that a number of amino acids change their environment during photoconversion of Pr to Pfr, which can be reversed by subsequent photoconversion back to Pr. Through three-dimensional NMR spectroscopy before and after light photoexcitation, it should now be possible to define the movements of the chromophore and binding pocket during photoconversion. We also generated a series of strongly red fluorescent derivatives of SyB-Cph1, which based on their small size and thermostability may be useful as cell biological reporters.     

Integral association of phytochrome with a membranous fraction from etiolatedAvena shoots: red/far-red photoreversibility and in vitro characterization

The results reported in this paper provide strong evidence to support the belief that the small percentage of phytochrome recovered in low-speed centrifugation pellets, when prepared in the absence of divalent cations after various in vivo irradiations, is not simply a manifestation of non-specific co-precipitation of soluble phytochrome.The far-red reversibility of the observed near-doubling of phytochrome pelletability after in vivo red irradiation indicates that phytochrome pelletability in the absence of divalent cations is a phytochrome-controlled response. The characteristics of the pelleted phytochrome indicate a strong, hydrophobic interaction with membranes. A tentative proposal to explain the observed characteristics of the association of phytochrome with membranous material in the absence of divalent cations after different in vivo irradiations has been put forward.Abbreviations Pfr phytochrome in the far-red light absorbing form - Pr phytochrome in the fat-red light absorbing form - Ptot total phytochrome - R red light irradiation - FR far-red light irradiation     

Phytochrome-membrane interactions as a factor in stomatal opening

Red light enhances stomatal opening in Commelina communis L. This light effect is reversed by far-red irradiation. Pretreatment with filipin, which competitively inhibits phytochrome binding to membranes, also inhibits light-enhanced opening. The pretreatment with filipin is more inhibitory if preceded by red irradiation, than after far-red irradiation. Similar results are obtained with cycloheximide and low temperature, which retard phytochrome synthesis more than its degradation. This result may indicate an enhanced release of phytochrome in the Pfr form from binding sites rather than release of phytochrome in the Pr form. This points towards the possibility that phytochrome degradation and its release from binding sites are coupled.     

Spectral properties of soluble and pelletable phytochrome from epicotyls of etiolated pea seedlings

The red-light(R)-absorbing form of phytochrome (Pr) was detected spectrophotometrically in a 20,000 g particulate fraction prepared from a 1,000 g supernatant fraction from epicotyl tissue of pea (Pisum sativum L.) seedlings grown in the dark and only briefly exposed to dim green light. The difference spectrum of phytochrome in this fraction was essentially the same as that of soluble phytochrome from the same tissue. When the non-irradiated 20,000 g particulate fraction was incubated in the dark at 25° C, an absorbance change (decrease) of Pr after actinic red irradiation was found only in the far-red (FR) region. When the 20,000 g particulate fraction was irradiated with R and then incubated in the dark, the FR-absorbing form of phytochrome (Pfr) disappeared spectrally at a rate about half that in the soluble fraction, and the difference spectrum of the Pr which became detectable after dark incubation of the 20,000 g particulate fraction was markedly distorted. In contrast, Pfr in a 20,000 g particulate fraction prepared from tissues irradiated with R did not change optically during dark incubation at 25° C for 60 min, while Pfr in the soluble fraction from the same tissue disappeared in the dark. No dissociation of either Pr or Pfr from the 20,000 g particulate fraction was indicated during a 60-min dark incubation at 25° C, but Pfr in a 20,000 g particulate fraction prepared in vitro from R-irradiated 1,000 g supernatant fraction in the presence of CaCl₂ disappeared spectrally and the difference spectrum of Pr in the 20,000 g particulate fraction became quite distorted during the dark incubation.Abbreviations Pr red-light-absorbing form of phytochrome - Pfr far-red-light-absorbing form of phytochrome - FR far-red light - FR1 first actinic far-red light - FR2 second actinic far-red light - R red light - R1 first actinic red light - 1kS 1,000 g supernatant fraction - 20kS 20,000 g supernatant fraction - 20kP 20,000 g particulate fraction     

Differences in the physical properties of native and partially degraded phytochrome as probed by their differential sensitivity to permanganate oxidation

The differential sensitivities to permanganate oxidation of the red and far-red forms of native phytochrome from Avena sativa L. cv Mulaga (isolated as Pfr from red-irradiated tissue) and of partially degraded phytochrome (isolated as Pr from nonirradiated tissue) were determined. The far-red absorbing form of partially degraded phytochrome was 5 times more sensitive than its red-absorbing form, while both the far-red and red forms of native phytochrome exhibited identical sensitivity. The present data obtained with partially degraded phytochrome are in apparent agreement with the data and model of Hahn, Kang, and Song (1980 Biochem Biophys Res Commun 97: 1317-1323). Their model suggests that the chromophore of the red-absorbing form of phytochrome is buried in a hydrophobic crevice in the protein, while that of the far-red form is exposed. The data obtained with native phytochrome, however, are at variance with their model. Our data obtained with native phytochrome suggests that the chromophore of the red and the far-red absorbing forms of native phytochrome both are in a relatively protected environment and that only following partial proteolytic degradation of the phytochrome does the chromophore of its far-red form become relatively more exposed. The protective influence of the labile peptide could either be direct, because of its close physical proximity to the chromophore, or indirect, resulting in an alteration in chromophore-protein interaction.     

Discrimination between the red- and far-red-absorbing forms of phytochrome from Avena sativa L. by monoclonal antibodies

A set of rat monoclonal antibodies (ARC MAC 48 to 52 and 54 to 56), raised to phytochrome from dark-grown seedlings of Avena sativa L. was tested for the ability to discriminate between the red-absorbing (Pr) and far-red-absorbing (Pfr) forms of phytochrome by indirect enzyme-linked immunosorbent assay. MAC 50 bound more strongly to Pfr and MAC 49 and 52 showed preferential binding to Pr from extracts of dark-grown Avena seedlings; MAC 50 also bound more strongly to Pfr from brushite-purified phytochrome. The remainder of the monoclonal antibodies and a rabbit polyclonal antiphytochrome preparation did not discriminate between Pr and Pfr. The results provide evidence for conformational changes in defined regions of the phytochrome apoprotein upon photoconversion.Abbreviations ELISA enzyme-linked immunosorbent assay - FR far-red light - McAb monoclonal antibody(ies) - PBS phosphate-buffered saline - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light - PMSF phenylmethylsulphonylfluoride     

Phytochrome in Pharbitis nil during and after de-etiolation

Phytochrome (P) was measured by in vivo spectrophotometry in the cotyledons of Pharbitis nil Choisy cv. Violet. In etiolated plants exposure to red light (R) results in a rapid fall in P content by destruction of the far-red absorbing form of phytochrome (Pfr). In continuous R or white light the P content falls as a result of destruction to a stable 3% of that originally present. In Norflurazon-treated white light de-etiolated plants, Pfr undergoes reversion in darkness to the red absorbing form of phytochrome (Pr) with a half life of 4 h at 19°C, while total P remains constant. Synthesis of Pr after de-etiolation occurs at a slow rate and is enhanced by terminating the de-etiolation light treatment with far-red light. The results are discussed in relation to the P control of flowering.     

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     

Experimentally induced binding of phytochrome to mitochondrial and microsomal fractions in etiolated pea shoots

Summary A brief irradiation with red light of pea (Pisum sativum L.) shoot segments kept at 0° resulted in very rapid binding of both Pr and Pfr to mitochondrial and microsomal fractions. The effect was not far-red reversible. The amount of phytochrome bound to the mitochondrial fraction was proportional to the percentage of Pfr of the fraction, and the ratio of Pr and Pfr in the bound form was the same as that in 12,000 x g supernatant. After a brief exposure of the segments to red light at 0° and a subsequent dark incubation at 30° in Tris-HCL buffer containing dithiothreitol or EDTA, which bot inhibit Pfr decay, the contents of phytochrome in the mitochondrial and microsomal fractions were significantly enhanced with time. The red-light effect was reversed by far-red light. The increase of the phytochrome content in the particulate fractions continued for at least 2 h, reaching a ca. 3 times higher level in terms of (A) per mg protein.Abbreviations R red - FR far-red - Pr red-absorbing form of phytochrome - Pfr far-red-absorbing form of phytochrome