Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
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
References
Bürk, R.R., Eschenbruch, M., Leuthard, P., Steck, G. (1983) Sensitive detection of proteins and peptides in polyacrylamide gels after formaldehyde fixation. Methods Enzymol. 91, 247–254
Grimm, R., Rüdiger, W. (1986) A simple and rapid method for isolation of 124 kDa oat phytochrome. Z. Naturforsch. 41 c, 988–992
Grimm, R., Gast, D., Rüdiger, W. (1989) Characterization of a protein-kinase activity associated with phytochrome from etiolated oat (Avena sativa L.) seedlings. Planta 178, 199–206
Gross, J., Seyfried, M., Fukshansky, L., Schäfer, E. (1984) In vivo spectrophotometry. In: Techniques in photomorphogenesis, pp. 131–158, Smith, H., Holmes, M.G., eds. Academic Press, London
Hershey, H.P., Barker, R.F., Idler, K.B., Lissemore, J.L., Quail, P.H. (1985) Analysis of cloned cDNA and genomic sequences for phytochrome: Complete amino acid sequences for two gene products expressed in etiolated Avena. Nucleic Acids Res. 13, 8543–8560
Jabben, M., Shanklin, J., Vierstra, R.D. (1989) Ubiquitin-phytochrome conjugates: Pool dynamics during in vivo phytochrome degradation. J. Biol. Chem. 264, 4998–5005
Kay, S.A., Keith, B., Shinozaki, K., Chye, M.-L., Chua, N.-H. (1989) The rice phytochrome gene: structure, autoregulated expression, and binding of GT-1 to a conserved site in the 5′ upstream region. Plant Cell 1, 351–360
Kim, I.S., Bai, U., Song, P.S. (1989) A purified 124-kDa oat phytochrome does not possess a protein kinase activity. Photochem. Photobiol. 49, 319–323
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685
Larson, E., Howlett, B., Jagendorf, A. (1986) Artificial reductant enhancement of the Lowry Method for protein determination. Anal. Biochem. 155, 243–248
Laskey, R.A., Mills, A.D. (1975) Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335–341
Lissemore, J.L., Quail, P.H. (1988) Rapid trancriptional regulation by phytochrome of the genes for phytochrome and chlorophyll a/b-binding protein in Avena sativa. Mol. Cell Biol. 8, 4840–4850
Lissemore, J.L., Colbert, J.T., Quail, P.H. (1987) Cloning of cDNA for phytochrome from etiolated Cucurbita and coordinate photoregulation of the abundance of two distinct phytochrome transcripts. Plant Mol. Biol. 8, 485–496
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275
Mackenzie, J.M., Coleman, R.A., Briggs, W.R., Pratt, L.H. (1975) Reversible redistribution of phytochrome within the cell upon conversion to its physiologically active form. Proc. Natl. Acad. Sci. USA 72, 799–803
McCurdy, D.W., Pratt, L.H. (1986a) Kinetics of intracellular redistribution of phytochrome in Avena coleoptiles after its photoconversion to the active, far-red-absorbing form. Planta 167, 330–336
McCurdy, D.W., Pratt, L.H. (1986b) Immunogold electron microscopy of phytochrome in Avena: Identification of intracellular sites responsible for phytochrome sequestering and enhanced pelletability. J. Cell Biol. 103, 2541–2550
Mohr, H., Shropshire, W. Jr. (1983) An introduction to photomorphogenesis for the general reader. In: Encyclopedia of plant physiology vol. 16A: Photomorphogenesis, pp. 24–38, Shropshire, W. Jr., Mohr, H. eds. Springer, Berlin Heidelberg New York
Neville, D.M. Jr. (1971) Molecular weight determination of proteindodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J. Biol. Chem. 246, 6328–6334
Pratt, L.H., Marmé, D. (1976) Red-light-enhanced phytochrome pelletability. Re-examination and further characterization. Plant Physiol. 58, 686–692
Quail, P.H. (1978a) Irradiation-enhanced phytochrome pelletability in Avena: In vivo development of a potential to pellet and the role of Mg2+ in its expression in vitro. Photochem. Photobiol. 27, 147–153
Quail, P.H. (1978b) Irradiation-enhanced phytochrome pelletability in Avena: Pigment release by Mg2+-gradient elution. Photochem. Photobiol. 27, 759–765
Quail, P.H., Marmé, D., Schäfer, E. (1973) Particle-bound phytochrome from maize and pumpkin. Nature New Biol. 245, 189–191
Sato, N. (1988) Nucleotide sequence and expression of the phytochrome gene in Pisum sativum: Differential regulation by light of multiple transcripts. Plant Mol. Biol. 11, 697–710
Schäfer, E. (1977) Kunstlicht und Pflanzenzucht. In: Optische Strahlungsquellen, pp. 249–266, Albrecht H., ed. Lexika, Grafenau
Schäfer, E. (1987) Primary action of phytochrome. In: Phytochrome and photoregulation in plants, pp. 279–289, Furuya, M., ed. Academic Press, London New York
Schägger, H., von Jagow, G. (1987) Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368–379
Speth, V., Otto, V., Schäfer, E. (1987) Intracellular localization of phytochrome and ubiquitin in red-light-irradiated oat coleoptiles by electron microscopy. Planta 171, 332–338
Tobin, E.M., Silverthorne, J. (1985) Light regulation of gene expression in higher plants. Annu. Rev. Plant Physiol. 36, 569–593
Vierstra, R.D., Quail, P.H. (1986) Phytochrome. The protein. In: Photomorphogenesis in plants, 35–60, Kendrick, R.E., Kronenberg, G.H.M., eds. Nijhoff, Dordrecht
Vigil, E.L. (1970) Cytochemical and developmental changes in microbodies (glyoxysomes) and related organelles of castor bean endosperm. J. Cell Biol. 46, 435–454
Wong, Y.-S., Cheng, H.-C., Walsh, D.A., Lagarias, J.C. (1986) Phosphorylation of Avena phytochrome in vitro as a probe of light-induced conformational changes. J. Biol. Chem. 261, 12089–12097
Yamamoto, K.T., Smith, W.O. Jr., Furuya, M. (1980) Photoreversible Ca2+-dependent aggregation of purified phytochrome from etiolated pea and rye seedlings. Photochem. Photobiol. 32, 233–239
Author information
Authors and Affiliations
Additional information
This work was supported by Deutsche Forschungsgemeinschaft. The competent technical assistance of Karin Fischer is gratefully acknowledged.
Rights and permissions
About this article
Cite this article
Hofmann, E., Grimm, R., Harter, K. et al. Partial purification of sequestered particles of phytochrome from oat (Avenu sativa L.) seedlings. Planta 183, 265–273 (1991). https://doi.org/10.1007/BF00197798
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00197798