Abstract
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.
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Abbreviations
- 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
- λ Rmax , λ FRmax :
-
maxima of the phototransformation difference spectrum in the red and far-red region
References
Bolton, G.W., Quail, P.H. (1981) A method for preparing plant tissue extracts for spectrophotometric measurement of phytochrome. (Abstr.) Plant Physiol. 67, Suppl, 104
Bolton, G.W., Quail, P.H. (1982) Cell-free synthesis of phytochrome apoprotein. Planta 155, 212–217
Burnette, W.N. (1981) “Western blotting”: Electrophoretic transfer of proteins from SDS-PAGE to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein. Anal. Biochem. 112, 195–203
Butler, W.L., Norris, K.H. (1960) The spectrophotometry of dense light scattering material. Arch. Biochem. Biophys. 87, 31–40
Butler, W.L., Lane, H.C., Siegelman, H.W. (1963) Nonphotochemical transformations of phytochrome in vivo. Plant Physiol. 38, 514–519
Cleveland, D.W. (1983) Peptide mapping in one dimension by limited proteolysis of sodium dodecyl sulfate-solubilized proteins. Methods Enzymol. 96, 222–229
Colbert, J.T., Hershey, H.P., Quail, P.H. (1983) Autoregulatory control of translatable phytochrome mRNA levels. Proc. Natl. Acad. Sci. USA 80, 2248–2252
Daniels, S.M., Quail, P.H. (1984) Monoclonal antibodies to three separate domains on 124 kilodalton phytochrome from Avena. Plant Physiol., 76, 622–626
Gottman, K., Schäfer, E. (1983) Analysis of phytochrome kinetics in light-grown Avena sativa L. seedlings. Planta 157, 392–400
Hillman, W.S. (1967) The physiology of phytochrome. Annu. Rev. Plant Physiol. 18, 301–324
Hunt, R.E., Pratt, L.H. (1979) Phytochrome immunoaffinity purification. Plant Physiol. 64, 332–336
Jabben, M. (1980) The phytochrome system in light-grown Zea mays L. Planta 149, 91–96
Jabben, M., Holmes, M.G. (1983) Phytochrome in light-grown plants. In: Encyclopedia of plant physiology, N.S., vol. 16B: Photomorphogenesis, pp. 704–722, Shropshire, W., Jr., Mohr, H., eds. Springer, Berlin Heidelberg New York Tokyo
Jabben, M., Deitzer, G.F. (1978) Spectrophotometric phytochrome measurements in light-grown Avena sativa L. Planta 143, 309–313
Jendrisak, J.J., Burgess, R.R. (1975) A new method for the large-scale purification of wheat-germ DNA-dependant RNA polymerase II. Biochemistry 14, 4639–4645
Jose, A.M., Vince-Prue, D., Hilton, J.R. (1977) Chlorophyll interference with with phytochrome measurement. Planta 135, 119–123
Kessler, S.W. (1976) Cell membrane antigen isolation with the Staphylococcal protein A-antibody adsorbent. J. Immunol. 117, 1482–1490
Knecht, D.A., Dimond, R.L. (1984) Visualization of antigenic proteins on western blots. Anal. Biochem. 136, 180–184
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685
Lane, H.C., Siegelman, H.W., Butler, W.L., Firer, E.M. (1963) Detection of phytochrome in green plants. Plant Physiol. 38, 414–416
Litts, J.C., Kelley, J.M., Lagarias, J.C. (1983) Structure-function studies on phytochrome. J. Biol. Chem. 258, 11025–11031
Leary, J.J., Brigati, D.J., Ward, D.C. (1983) Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bioblots. Proc. Natl. Acad. Sci. USA 80, 4045–4049
Lowry, O., Rosenbrough, N., Farr, A., Randall, R. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275
Pike, C.S., Briggs, W.R. (1972a) The dark reactions of rye phytochrome in vivo and in vitro. Plant Physiol. 49, 514–520
Pike, C.S., Briggs, W.R. (1972b) Partial purification and characterization of a phytochrome-degrading neutral protease from etiolated oat shoots. Plant Physiol. 49, 521–530
Pratt, L.H. (1982) Phytochrome: the protein moiety. Annu. Rev. Plant Physiol. 33, 557–582
Sartorelli, A.C., Fischer, D.S., Downs, W.G. (1966) The use of Sarcoma 180/TG to prepare hyperimmune ascitic fluid in the mouse. J. Immunol. 96, 676–682
Shimazaki, Y., Cordonnier, M., Pratt, L.H. (1983) Phytochrome quantitation in crude extracts of Avena by enzyme-linked immunosorbent assay with monoclonal antibodies. Planta 159, 534–544
Shropshire, W., Jr., Mohr, H. (1983) Photomorphogenesis. Springer, Berlin Heidelberg New York Tokyo
Siegelman, H.W., Butler, W.L. (1965) Properties of phytochrome. Annu. Rev. Plant Physiol. 16, 383–392
Smith, W.O. (1983) Phytochrome as a molecule. In: Encyclopedia of plant physiology, N.S., vol. 16A: Photomorphogenesis, pp. 96–118, Shropshire, W., Jr., Mohr, H., eds. Springer, Berlin Heidelberg New York Tokyo
Taylor, A.O., Bonner, B.A. (1967) Isolation of phytochrome from the alga Mesotaenium and the liverwort Sphaerocarpus. Plant Physiol. 42, 762–766
Thomas, B., Crook, N.E., Penn, S.E. (1984) An enzyme-linked immunosorbent assay for phytochrome. Physiol. Plant. 60, 409–415
Tokuhisa, J.G., Quail, P.H. (1983) Spectral and immunochemical characterization of phytochrome isolated from light-grown Avena sativa. (Abstr.) Plant Physiol. 72, Suppl., 85
Tokuhisa, J.G., Daniels, S.M., Quail, P.H. (1984) Two distinct molecular species of phytochrome in light-grown Avena sativa. (Abstr.) Plant Physiol. 75, Suppl., 73
Vernon, L.P. (1960) Spectrophotometric determination of chlorophylls and phaeophytins in plant extracts. Anal. Chem. 32, 1144–1150
Vierstra, R.D., Quail, P.H. (1982a) 124 kilodalton phytochrome. Proc. Natl. Acad. Sci. USA 79, 5272–5276
Vierstra, R.D., Quail, P.H. (1982b) Proteolysis alters the spectral properties of 124 kdalton phytochrome from Avena. Planta 156, 158–165
Vierstra, R.D., Quail, P.H. (1983) Purification and initial characterization of 124-kilodalton phytochrome from Avena. Biochemistry 22, 2498–2505
Vierstra, R.D., Tokuhisa, J.G., Quail, P.H. (1983) A solid-phase antibody approach to the subcellular localization of phytochrome. In: Current Topics in Plant Biochemistry and Physiology, vol. 1, pp. 37–50, Randall, D.D., Blevins, D.G., Larson, R., eds. University of Missouri, Columbia, USA
Vierstra, R.D., Cordonnier, M.-M., Pratt, L.H., Quail, P.H. (1984) Native phytochrome: Immunoblot analysis of relative molecular mass and in-vitro proteolytic degradation for several plant species. Planta 160, 521–528
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Tokuhisa, J.G., Daniels, S.M. & Quail, P.H. Phytochrome in green tissue: Spectral and immunochemical evidence for two distinct molecular species of phytochrome in light-grown Avena sativa L.. Planta 164, 321–332 (1985). https://doi.org/10.1007/BF00402943
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DOI: https://doi.org/10.1007/BF00402943