Characterization of a protein-kinase activity associated with phytochrome from etiolated oat (Avena sativa L.) seedlings

A protein-kinase activity which is co-purified with phytochrome from etiolated oat seedlings was investigated in some detail. Whereas phytochrome was always phosphorylated in solution (together with some contaminating protein bands), radioactive phosphate was not found in the phytochrome band after native gel electrophoresis and incubation of the entire gel with labeled ATP. Since protein kinases are usually autophosphorylated under these conditions, the result shows that the kinase activity does not reside in the phytochrome molecule itself. Radioactivity was exclusively detected in a band with the apparent molecular weight 450 kDa; sodium-dodecyl-sulfate gel electrophoresis revealed an apparent molecular weight of 60 kDa for the phosphorylated subunit. The N-terminal amino-acid sequence A L E S _A ^G K _Q ^L V P W was determined for this subunit which is a potential candidate for the protein kinase. The optimum conditions (pH, metal ion concentration) and kinetics of the phosphorylation reaction were determined. The presumed connection between proteinkinase activity and the signal chain leading from the far-red-absorbing form of phytochrome to physiological responses still awaits elucidation.Abbreviations Bistris 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol - kDa kilodalton - Pfr far-red absorbing form of phytochrome - Pr red-absorbing form of phytochrome - PMBS p-chloromercuribenzenesulfonate - SDS sodium dodecyl sulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol Dedicated to Professor A. Trebst on the occasion of his 60th birthday     

The levels of two distinct species of phytochrome are regulated differently during germination in Avena sativa L.

The abundance and molecular mass of phytochrome in germinating embryos of A. sativa (oat) grown in light or darkness have been monitored using immunoblot and spectrophotometric assays. Immunoblot analysis shows that imbibed but quiescent embryos have two immunochemically distinct species of phytochrome with monomeric molecular masses of 124 and 118 kDa (kdalton). The 118-kDa species has the properties of the 118-kDa phytochrome extracted from fully green oat tissue (J.G. Tokuhisa, S.M. Daniels, P.H. Quail, 1985, Planta 164, 321–332), whereas the 124-kDa polypeptide appears similar to the well-characterized photoreceptor of etiolated tissue. The capacity of antibodies directed against etiolated-oat phytochrome to immunoprecipitate the 124-kDa species but not the 118-kDa species has been exploited to quantitate the levels of each separately over a 72-h time course of germination and seedling development. The abundance of the 124-kDa molecule increases at least 200-fold in etiolated seedlings over 72 h whereas in light-grown seedlings the level of this molecule is relatively constant. In contrast, the amount of the 118-kDa species increases only twofold in both dark- and light-grown seedlings over the same period of time. These data indicate that whereas the abundance of 124-kDa phytochrome is regulated at the protein level by the well-documented, differential stability of the red- and far-red-absorbing forms in vivo, the 118-kDa molecule is present at a low constitutive level, presumably reflecting no such difference in the stability of the two spectral forms.Abbreviations ELISA enzyme-linked immunosorbent assay - Ig immunoglobulin - kDa kilodalton - Pfr, Pr far-red-absorbing and red-absorbing forms of phytochrome, respectively - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

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 — all regions marked by a set of monoclonal antibodies reflect conformational changes

Monoclonal antibodies to defined locations on six regions of the phytochrome molecule (from Avena sativa L. or Zea mays L.) were each found to have a different affinity toward the farred-absorbing form of phytochrome (Pfr) and the red-absorbing form (Pr). The differences were small, but were consistently shown by antibodies which bind to the vicinity of the aminoterminus, the carboxylterminus and to sequences in between. It seems that the conformational differences between Pr and Pfr extend over the whole molecule in as far as it is represented by these regions and the antibodies binding to them.Abbreviations Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome     

Phytochrome structure: Peptide fragments from the amino-terminal domain involved in protein-chromophore interactions

We have undertaken a study of the structure of the amino-terminal domain of the phytochrome polypeptide purified from Avena sativa L. Amino-acid sequencing was used to indentify arginine 52 as the precise location of a conformation-specific cleavage of phytochrome by subtilisin. The location of the epitopes for a class of monoclonal antibodies designated type 2 has been shown to be located between approx. 10 and 20 kilodaltons (kDa) from the amino terminus. These two new spatial markers, in addition to the chromophore and another epitope recognized by type 1 monoclonal antibodies and located within 6 kDa from the amino terminus, have been used to map the locations of several new protease-accessible sites along the polypeptide. After extensive digestion of phytochrome with subtilisin, a stable spectrally-active group of peptides remains. Within this group is a 16-kDa chromopeptide which, either alone or as part of an assemblage of peptides, elutes from a size-exclusion column under nondenaturing conditions at a volume consistent with a molecular mass of 35–40 kDa. This group of peptides has an absorbance spectrum similar to the red-absorbing form of phytochrome (Pr) and is red/far-red photoreversible between this and a photobleached form. These data indicate that this group of peptides still retains the principal structural requisites for Pr-chromophore-protein interactions and for photoreversibility, but not for Pfr (far-red-absorbing phytochrome)-chromophore-protein interactions. It is uncertain if these structural requisites reside exclusively on the 16-kDa chromopeptide or result from an assemblage of these peptides. However, we have excluded any role for an adjacent 14-kDa fragment (approximately residues 50 to 200) in the observed spectral properties since it can be selectively removed without any effect on the photoreversibility.Abbreviations Da dalton - M_r relative molecular mass - Pr, Pfr red and far-red-absorbing forms of phytochrome, respectively - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis This work was presented, in part, at the XVI Yamada Conference on Phytochrome and Plant Photomorphogenesis, Okazaki, Japan, October 1986     

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     

Changes in topography and function of thylakoid membranes following membrane protein phosphorylation

The kinetics of the intracellular redistribution of phytochrome (sequestering) in Avena sativa L. coleoptiles following a brief, saturating actinic pulse of red (R) light have been determined. Immunocytochemical labelling of phytochrome with monoclonal antibodies showed that at 22°C sequestering can occur within 1–2 s from the onset of R irradiation and is dependent upon the continued presence of the far-red-absorbing form of phytochrome (Pfr). The initial rate, but not the final extent, of sequestering is reduced by lowering the temperature of the tissue to 1°C. Sequestering at 22°C appears to involve two distinct stages: (1) a rapid association of Pfr with putative binding sites initiates the sequestered condition, following which (2) these sites of sequestered phytochrome appear to aggregate. Neither of these two processes was affected by the cytoskeletal inhibitors colchicine or cytochalasin B. Phytochrome sequestering therefore resembles R-light-induced phytochrome pelletability with respect to kinetics, temperature sensitivity, and dependence upon the continued presence of Pfr in the cell.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DIC differential interference contrast - FR far-red - Ig immunoglobulin - Pfr, Pr far-red-absorbing and red-absorbing form of phytochrome, respectively - R red     

The role of separate molecular domains in the structure of phytochrome from etiolated Avena sativa L.

The spectral properties of peptides generated from etiolated-Avana, 124-kDa (kilodalton) phytochrome by endogenous protease(s) have been studied to assess the role of the amino-terminal and the carboxyl-terminal domains in maintaining the proper interaction between protein and chromophore. The amino-terminal, 74-kDa chromopeptide, a degradation product of the far-red absorbing form of the pigment (Pfr), is shown to be spectrally similar to the 124-kDa, undegraded molecule. The minimum and maximum of the difference spectrum (Pr-Pfr) are 730 and 665 nm, respectively, and the spectral-change ratio is unity. Also, like undegraded, 124-kDa phytochrome, the 74-kDa peptide exhibits minimal dark reversion. These data indicate that the 55-kDa, carboxyl-terminal half of the polypeptide does not interact with the chromophore and may not have a role in the structureal integrity of the amino-terminal domain. The 64-kDa chromopeptide can be generated directly from the 74-kDa species by cleavage of 10 kDa from the amino terminus upon incubation of this species as Pr. Accompanying this conversion are changes in the spectral properties, namely, a shift in the difference spectrum minimum to 722–724 nm and a tenfold increase in the capacity for dark reversion. These data indicate that the 6–10 kDa, amino-terminal segment continues to function in its role of maintaining proper chromophore-protein interactions in the 74-kDa peptide as it does in the undegraded molecule. Conversely, removal of this segment upon proteolysis to the 63-kDa species leads to aberrant spectral properties analogous to those observed when this domain is lost from the full-length, 124-kDa molecule, resulting in the 118/114-kDa degradation products. The data also show that photoconversion of the 74-kDa chromopeptide from Pfr to Pr exposes proteolytically susceptible sites in the same way as in the 124-kDa molecule. Thus, the separated, 74-kDa amino-terminal domain undergoes a photoinducible conformational change comparable to that in the intact molecule.Abbreviations and symbols Da dalton - Pfr far-red-absorbing from of phytochrome - PMSF phenylmethylsulfonyl fluoride - Pr red-absorbing form of phytochrome - R red light - FR lar-red light - A _r/A _fr spectral change ratio - _max ^FR peak maximum (nm) of Pfr absorbance     

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     

Polyclonal antibodies raised to phycocyanins contain components specific for the red-absorbing form of phytochrome

Polyclonal antibodies raised in rabbits to a mixture of sodium-dodecyl-sulphate-denatured C- and allo-phycocyanin, isolated from Anabaena cylindrica, cross-react with 124-kilodalton (kDa) phytochrome from etiolated oats, in enzyme-linked immunosorbent assays and on Western blots. The component(s) of the anti-phycocyanin serum that cross-reacts with phytochrome appears to be specific for the red-absorbing form of phytochrome (Pr). These antibodies can be detached from Pr by irradiation with red light, and thus show photoreversible binding. This property has been used to immunopurify the anti-phytochrome component from the antiserum using red light as the eluting agent. Competition assays and epitope-mapping studies indicate that the anti-phytochrome component may bind to a site located between 6 and 10 kDa from the amino-terminus of etiolated oat phytochrome.Abbreviations ELISA enzyme-linked immunosorbent assay - kDa kilodaton - FR far-red light - Pfr far-red-light-absorbing form of phytochrome - Pr red-light-absorbing form of phytochrome - R red light - SDS sodium dodecyl sulphate     

Interaction of light and temperature on seed germination of Rumex obtusifolius L.

Germination of Rumex obtusifolius L. seeds (nutlets) is low in darkness at 25° C. Germination is stimulated by exposure to 10 min red light (R) and also by a 10-min elevation of temperature to 35° C. A 10-min exposure to far-red light (FR) can reverse the effect of both R (indicating phytochrome control) and 35° C treatment. Fluence-response curves for this reversal of the effect of R and 35° C treatments are quantitatively identical. Treatment for 10 min with light of wavelenght 680, 700, 710 and 730 nm, after R and 35° C treatment, demonstrates that germination induced by 35° C treatment results from increased sensitivity to a pre-existing, active, far-red-absorbing form of phytochrome (Pfr) in the seeds.Abbreviations FR far-red light - P phytochrome - Pr red-absorbing form of P - Pfr far-red-absorbing form of P - R red light     

Phytochrome Modification and Light-enhanced, In Vivo-induced Phytochrome Pelletability

Phytochrome that was induced by red irradiation in vivo to pellet with subcellular material and that was released from the pellet by removal of divalent cations exhibited altered characteristics. Compared to phytochrome extracted in a soluble red-absorbing form from etiolated tissue, pelleted and released phytochrome, which was also assayed in the red-absorbing form even though pelleted in the far-red-absorbing form, showed 50% greater micro complement fixation activity, eluted closer to the void volume of a Sephadex G-200 column, and electrophoresed more slowly on sodium dodecyl sulfate-polyacrylamide gels. Data presented here document that phytochrome pelleted in the far-red-absorbing form differs from soluble phytochrome extracted from nonirradiated tissue. These data, however, do not permit the conclusion that there is a causal relationship between pelletability and phytochrome modification.     

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)     

Cell-free synthesis of phytochrome apoprotein

A single polypeptide is immunospecifically precipitated by monospecific antiphytochrome from the total translation products of both wheat-germ and rabbit-reticulocyte cell-free protein synthesizing systems programmed with oat (Avena sativa L.) poly(A) RNA. The mobility of this polypeptide is slightly lower on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis than that of immunoaffinity-purified, 118 kdalton phytochrome and corresponds to an apparent molecular weight of 124 kdalton. Evidence against the possibility that this mobility difference results from intracellular processing of the 124-kdalton protein is provided by extraction of freeze-dried tissue directly into boiling SDS-containing buffer. This procedure yields a phytochrome species with a mobility on SDS polyacrylamide gel electrophoresis indistinguishable from that of the in-vitro translation product. Together the data indicate that the phytochrome polypeptide is synthesized in its mature form in the cell but is subject to modification to a form with lower apparent molecular weight during immunopurification.Abbreviations IgG immunoglobulin G - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Large-scale partial purification of phytochrome from green leaves of Avena sativa L.

Phytochrome from 10 or 11-d-old oat (Avena sativa L. cv. Garry) leaves, which were harvested just prior to sunset from plants grown in a greenhouse in the absence of supplemental illumination, was purified an estimated 250-fold by sequential poly(ethylenimine) and ammonium-sulfate fractionations, followed by linear-gradient hydroxyapatite chromatography. Compared to earlier protocols, the one presented here is substantially more rapid, provides improved yield and purity, can be used with larger quantities of tissue, and eliminates an apparently immunodominant contaminant with a molecular mass of about 115 kDa (kilodalton). Phytochrome obtained by this procedure has an apparent monomer size of 123 kDa as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is estimated to be 0.6% pure. This purity permitted spectral analysis at wavelengths below 500 nm, in which region phytochromes from green and etiolated oat shoots do not differ markedly, as they do at longer wavelengths.Abbreviations Da Dalton - HA hydroxyapatite - Pfr, Pr farredand red-absorbing form of phytochrome, respectively - SDS sodium dodecyl sulfate This research was supported by the U.S. Department of Energy (contract DE-AC-09-81SR10925 to L.H.P.). The excellent technical assistance of Mrs. Donna Tucker and Mrs. Danielle Neal is gratefully acknowledged.     

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     

Immunochemical detection with rabbit polyclonal and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green Avena shoots

While two monoclonal antibodies directed to phytochrome from etiolated oat (Avena sativa L.) shoots can precipitate up to about 30% of the photoreversible phytochrome isolated from green oat shoots, most precipitate little or none at all. These results are consistent with a report by J.G. Tokuhisa and P.H. Quail (1983, Plant Physiol. 72, Suppl., 85), according to which polyclonal rabbit antibodies directed to phytochrome from etiolated oat shoots bind only a small fraction of the phytochrome obtained from green oat shoots. The immunoprecipitation data reported here indicate that essentially all phytochrome isolated from green oat shoots is distinct from that obtained from etiolated oat shoots. The data indicate further that phytochrome from green oat shoots might itself be composed of two or more immunochemically distinct populations, each of which is distinct from phytochrome from etiolated shoots. Phytochrome isolated from light-grown, but norflurazon-bleached oat shoots is like that isolated from green oat shoots. When light-grown, green oat seedlings are kept in darkness for 48 h, however, much, if not all, of the phytochrome that reaccumulates is like that from etiolated oat shoots. Neither modification during purification from green oat shoots of phytochrome like that from etiolated oat shoots, nor non-specific interference by substances in extracts of green oat shoots, can explain the inability of antibodies to recognize phytochrome isolated from green oat shoots. Immunopurified polyclonal rabbit antibodies to phytochrome from etiolated pea (Pisum sativum L.). shoots precipitate more than 95% of the photoreversible phytochrome obtained from etiolated pea shoots, while no more than 75% of the pigment is precipitated when phytochrome is isolated from green pea shoots. These data indicate in preliminary fashion that an immunochemically unique pool of phytochrome might also be present in extracts of green pea shoots.Abbreviation ELISA enzyme-linked immunosorbent assay - mU milliunit - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome     

Temperature- and seed-batch-related variation in the kinetic behaviour of phytochrome under ‘high-irradiance-response’ conditions

The characteristics of the high-irradiance response (HIR) of plant photomorphogenesis are thought to be the result of the interaction of both the light and dark reactions of phytochrome. Thus any variation in the rates of the dark reactions may be expected to lead to variation in the characteristics of the HIR. We report here substantial differences in the rates of the dark reactions between different seed batches of a single species (Sinapis alba L.), and also between different organs of seedlings from each of the batches of seed. Calculations of phytochrome dynamics from the measured dark-reaction rates show that the behaviour of Pfr under HIR conditions will vary considerably according to seed batch and seedling organ. Much larger differences in dark-reaction rates, and the resulting phytochrome dynamics, were found between 25° and 10° C. These lead to the prediction that the HIR will be much reduced at the lower temperature, and may be absent in some cases.Abbreviations and symbols HIR high-irradiance response - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - Ptot total phytochrome, Pr+Pfr - _ss Pfr/Ptot ratio which immediately establishes the phytochrome steady state     

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.     

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