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1.
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.  相似文献   

2.
Seven monoclonal antibodies (MAbs) have been prepared to phytochrome from green oat (Avena sativa L. cv. Garry) leaves. One of these MAbs (GO-1) cross-reacts with apoprotein of the phytochrome that is most abundant in etiolated oat shoots as assessed by immunoblot assay of fusion proteins expressed in Escherichia coli. The epitope for this MAb is located between amino acids 618 and 686 in the primary sequence of type 3 phytochrome (Hershey et al. 1985, Nucleic Acids Res. 13, 8543–8559), which is one of the predominant phytochromes in etiolated oats. Three other MAbs (GO-4, GO-5, GO-6) immunoprecipitate phytochrome isolated from green oat leaves, as evaluated by photoreversibility assay. GO-1, GO-4, GO-5 and GO-6 are therefore directed to phytochrome. While evidence obtained with the other three MAbs (GO-2, GO-7, GO-8) strongly indicates that they are also directed to phytochrome, this evidence is not as rigorous. Recognition of antigen by any of these seven MAbs is not significantly reduced by periodate oxidation, indicating that their epitopes probably do not include carbohydrate. All but GO-1 bind either very poorly or not at all the phytochrome that is abundant in etiolated oat shoots. These data reinforce earlier observations made with antibodies directed to phytochrome from etiolated oats, indicating (1) that the phytochromes that predominate in etiolated and green oats differ immunochemically and (2) that phytochrome preparations from green oat leaves contain very little of the phytochrome that is abundant in etiolated shoots. An hypothesis that these two immunochemically distinct phytochromes form heterodimers in vitroAbbreviations Da Dalton - DEAE diethylaminoethyl - ELISA enzyme-linked immunosorbent assay - HA hydroxyapatite - Ig immunoglobulin - MAb monoclonal antibody - SDS sodium dodecyl sulfate is supported by comparison of immunoblot data obtained with conventionally purified phytochrome from etiolated oats to that expressed as fusion protein in E. coli. This research was supported by the U.S. Department of Energy (contract DE-AC-09-81SR10925 to L.H.P.). We thank Dr. Lyle Crossland and Ms. Sue Kadwell for their assistance in the construction of the cDNA clones, and Dr. Gyorgy Bisztray for providing us with clone pCBP3712. Dr. Phillip Evans and Dr. Russell Malmberg kindly provided MAbs 4F3, 6F12 and 8C10, as well as a corresponding antigen preparation. The excellent technical assistance of Mrs. Donna Tucker and Mrs. Danielle Neal is gratefully acknowledged.  相似文献   

3.
Y. Shimazaki  L. H. Pratt 《Planta》1986,168(4):512-515
Thirty-nine antiserum preparations from eight rabbits were screened for their ability to precipitate the immunochemically distinct phytochrome that is obtained from green oat (Avena sativa L.) shoots. The antisera were obtained from rabbits immunized with either proteolytically degraded, but still photoreversible, 60-kDa (kilodalton) phytochrome, or approx. 120-kDa phytochrome, both of which were purified from etiolated oat shoots. The ability of these antisera to precipitate phytochrome from green oats was independent of the size of phytochrome used for immunization. While crude antisera immunoprecipitated as much as 80% of the phytochrome isolated from green oat shoots, antibodies immunopurified from these sera with a column of highly purified, approx. 120-kDa phytochrome from etiolated oats precipitated no more than about 5–10%.Abbreviations kDa kilodalton - mU milliunit  相似文献   

4.
Sequestered particles of phytochrome (SAPs) were partially purified from red-light-irradiated oat coleoptiles. Phytochrome pelletability was enhanced by using buffers containing 10 mM Mg2+ or high concentrations (0.6–0.8 M) of orthophosphate (Pi). Combining the pelletability of phytochrome in the presence of Mg2+ with that in the presence of 0.6 Pi resulted in a strong enrichment (about 100-fold) of pelletable phytochrome. Antisera were raised against Mg2+-Pi-pellets from darkgrown seedlings. Using these antisera, no evidence was found by Western blotting and immunocytochemistry that SAPs contain major proteins other than phytochrome. The major contamination of these enriched SAP preparations consisted of protein crystals which are probably catalase. The preparations contained methyltransferase and protein-kinase activities which were not associated with SAPs. Phytochrome purified from SAPs served as a substrate for protein-kinase activity but not for the methyltransferase activity. Phytochrome itself did not show any kinase activity.Abbreviations ME 2-mercaptoethanol - PAGE polyacrylamide gel electrophoresis - Pfr far-red-light-absorbing form of phytochrome - PMSF phenylmethylsulfonyl fluoride - SAP sequestered area of phytochrome - SDS sodium dodecyl sulfate This work was supported by Deutsche Forschungsgemeinschaft. The competent technical assistance of Karin Fischer is gratefully acknowledged.  相似文献   

5.
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  相似文献   

6.
The effect on the phytochrome system of light regimes establishing a range of photoequilibria was studied in two light grown dicotyledonous plants, both of which were treated with the herbicide SAN 9789 to prevent chlorophyll accumulation. In Sinapis alba L. cotyledons the results are comparable with phytochrome behaviour in etiolated mustard seedlings; the level of Pfr becomes independent of wave-length whereas the total phytochrome level is wave-length dependent. Contrasting properties are exhibited in Phaseolus aureus Roxb. leaves in which total phytochrome is unaffected by light quality; consequently the Pfr level is dependent on wavelength. Nevertheless, the amount of phytochrome in mung leaves increased after transfer to darkness suggesting that light still has a profound influence on the phytochrome system, even though light quality during the light period and prior to darkness does not.Abbreviations FR far-red light - WL white light - PAR photosynthetically active radiation - Pfr far-red light absorbing form of phytochrome - Pr red light absorbing form of phytochrome - Ptot total phytochrome level (=Pr+Pfr) - Pfr/Pfr+Pr - SAN 9789 4-chloro-5-(methylamino) 2(,, trifluoro-m tolyl)-3(2H)-pyridazinone  相似文献   

7.
8.
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  相似文献   

9.
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  相似文献   

10.
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  相似文献   

11.
Peter J. Watson  Harry Smith 《Planta》1982,154(2):121-127
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  相似文献   

12.
Proteolytic fragments were obtained by limited proteolysis of 124-kDa (kilodalton) phytochrome from etiolatedAvena sativa using trypsin, endoproteinase-Lys-C, endoproteinase-Glu-C and subtilisin. The fragments were separated by sodium dodecyl sulfate gel electrophoresis, blotted onto activated glass-fiber sheets and investigated by amino-acid sequencing in a gas-phase sequencer. Determination of N-terminal sequences in three to six Edman degradation steps allowed the exact localization of the fragments within the published entire amino-acid sequence of 124-kDaAvena phytochrome (H.P. Hershey, R.F. Barker, K.B. Idler, J.L. Lissemore, P.H. Quail (1985), Nucleic Acids Res.13, 8543–8559). From the knowledge of the exact sites for preferred proteolytic cleavage of undenatured phytochrome, conclusions on the conformation of the phytochrome protein were drawn. Sites of preferred cleavage are considered to be freely exposed to the environment whereas potential cleavage sites which are resistant to proteolysis over a long time are considered to be localized in the interior of the native phytochrome. Two different sites which are exposed in the far-red-absorbing form but not in the red-absorbing form of phytochrome are localized at amino-acid residues 354 and 753, respectively. The N-terminal region which is exposed only in the red-absorbing form stretches only as far as amino-acid residue 60.Abbreviations kDa kilodalton - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Dedicated to Professor W. Rau on the occasion of his 60th birthday.  相似文献   

13.
Y. Shimazaki  L. H. Pratt 《Planta》1985,164(3):333-344
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  相似文献   

14.
Michele Cope  Lee H. Pratt 《Planta》1992,188(1):115-122
The intracellular distribution of phytochrome in hypocotyl hooks of etiolated soybean (Glycine max L.) has been examined by immunofluorescence using a newly produced monoclonal antibody (Soy-1) directed to phytochrome purified from etiolated soybean shoots. Cortical cells in the hook region exhibit the strongest phytochrome-associated fluorescence, which is diffusely distributed throughout the cytosol in unirradiated, etiolated seedlings. A redistribution of immunocytochemically detectable hytochrome to discrete areas (sequestering) following irradiation with red light requires a few minutes at room temperature in soybean, whereas this redistribution is reversed rapidly following irradiation with far-red light. In contrast, sequestering in oat (Avena sativa L.) occurs within a few seconds (D. McCurdy and L. Pratt, 1986, Planta 167, 330–336) while its reversal by far-red light requires hours (J. M. Mackenzie Jr. et al., 1975, Proc. Natl. Acad. Sci. USA 72, 799–803). The time courses, however, of red-light-enhanced phytochrome pelletability and sequestering are similar for soybean as they are for oat. Thus, while these observations made with a dicotyledon are consistent with the previous conclusion derived from work with oat, namely that sequestering and enhanced pelletability are different manifestations of the same intracellular event, they are inconsistent with the hypothesis that either is a primary step in the mode of action of phytochrome.Abbreviations DIC differential interference contrast - FR far-red light - Ig immunoglobulin - Pfr, P far-red- and red-absorbing form of phytochrome, respectively - R red light This work was supported by National Science Foundation grant No. DCB-8703057.  相似文献   

15.
A method is described for the extraction of phytochrome from chlorophyllous shoots of Avena sativa L. Poly(ethyleneimine) and salt fractionation are used to reduce chlorophyll and to increase the phytochrome concentration sufficiently to permit spectral and immunochemical analyses. The phototransformation difference spectrum of this phytochrome is distinct from that of phytochrome from etiolated shoots in that the maximum in the red region of the difference spectrum is shifted about 15 nm to a shorter wavelength. Immunochemical probing of electroblotted proteins (Western blotting), using a method sensitive to 50 pg, demonstrates the presence of two polypeptides in green tissue that bind antiphytochrome antibodies: a predominant species with a relative molecular mass (Mr) of 118000 and a lesser-abundant 124000-Mr polypeptide. Under nondenaturing conditions all of the 124000-Mr species is immunoprecipitable, but the 118000-Mr species remains in the supernatant. Peptide mapping and immunochemical analysis with monoclonal antibodies show that the 118000-Mr species has structural features that differ from etiolated-oat phytochrome. Mixing experiments show that these structural differences are intrinsic to the molecular species from these two tissues rather than being the result of post-homogenization modifications or interfering substances in the green-tissue extracts. Together the data indicate that the phytochrome that predominates in green-tissue has a polypeptide distinct from the well-characterized molecule from etiolated tissue.Abbreviations and symbols Ig immunoglobulin - Mr relative molecular mass - Pfr, Pr far-red-absorbing and red-absorbing forms of phytochrome respectively - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - max R , max FR maxima of the phototransformation difference spectrum in the red and far-red region  相似文献   

16.
Phytochrome from leaves of light-grown oat (Avena sativa L. cv. Garry) plants is characterized with newly generated monoclonal antibodies (MAbs) directed to it. The results indicate that there are at least two phytochromes in green oat leaves, each of which differs from the phytochrome that is most abundant in etiolated oat tissue. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with reference to 124-kilodalton (kDa) phytochrome from etiolated oats, the two phytochromes from green oats have monomer sizes of 123 of 125 kDa. Immunoblot analysis of SDS, sample buffer extracts of lyophilized, green oat leaves indicates that neither the 125-kDa nor the 123-kDa polypeptide is a degradation product arising after tissue homogenization. Of the two, the 123-kDa phytochrome appears to be the predominant species in light-grown oat leaves. During SDS-PAGE in the presence of 1 mM Zn2+, 123-kDa phytochrome undergoes a mobility shift corresponding to an apparent mass increase of 2 kDa. In contrast, the electrophoretic mobility of 125-kDa phytochrome is unaffected by added Zn2+. Some MAbs that recognize 123-kDa phytochrome fail to recognize 125-kDa phytochrome and vice versa, indicating that these two phytochromes are not only immunochemically distinct from 124-kDa phytochrome, but also from each other. It is evident, therefore, that there are at least three phytochromes in an oat plant: 124-kDa phytochrome, which is most abundant in etiolated tissue, plus 123-and 125-kDa phytochromes, which predominate in light-grown tissue.Abbreviations Da Dalton - HA hydroxyapatite - MAb monoclonal antibody - PAb polyclonal antibody preparation - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate This research was supported by the U.S. Department of Energy (contract DE-AC-09-81SR10925 to L.H.P.). We thank Dr. Alan Jones, Department of Biology, University of North Carolina, Chapel Hill, USA, for kindly providing rabbit antiserum 4032, and Mrs. Donna Tucker and Mrs. Danielle Neal for their technical assistance.  相似文献   

17.
V. Speth  V. Otto  E. Schäfer 《Planta》1987,171(3):332-338
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  相似文献   

18.
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  相似文献   

19.
The effect of light on the phytochrome content of cauliflower (Brassica oleracea (L.) var. botrytis) curd was studied using in vivo spectrophotometry. It was found that light caused a rapid increase in phytochrome level whereas transfer to darkness caused a rapid loss, regardless of the amount of phytochrome initially present in the far red absorbing form. The amount of phytochrome detectable during continuous irradiation appears to be related to the photoequilibrium , and is thus controlled by phytochrome itself.Abbreviation Pr and Pfr red and far red absorbing forms of phytochrome, respectively  相似文献   

20.
E. Hofmann  V. Speth  E. Schäfer 《Planta》1990,180(3):372-377
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.  相似文献   

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