, Volume 178, Issue 2, pp 147–156 | Cite as

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

  • Alan M. Jones
  • Peter H. Quail


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.

Key words

Avena (phytochrome) Phytochrome (structure, photoreversibility) 





relative molecular mass

Pr, Pfr

red and far-red-absorbing forms of phytochrome, respectively


sodium dodecyl sulfate polyacrylamide gel electrophoresis


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Berkelman, T.R., Lagarias, J.C. (1986) Visualization of bilin-linked peptides and proteins in polyacrylamide gels. Anal. Biochem. 156, 194–201Google Scholar
  2. Daniels, S.M., Quail, P.H. (1984) Monoclonal antibodies to three separate domains on 124-kilodalton phytochrome from Avena. Plant Physiol. 76, 622–626Google Scholar
  3. Grimm, R., Lottspeich, F., Schneider, H.A.W., Rüdiger, W. (1986) Investigation of the peptide chain of 124 kDa phytochrome: Localization of proteolytic fragments and epitopes for monoclonal antibodies. Z. Naturforsch 41c, 83–90Google Scholar
  4. Grimm, R., Eckerskorn, C., Lottspeich, F., Zenger, C., Rüdiger, W. (1988) Sequence analysis of proteolytic fragments of 124-kilodalton phytochrome from etiolated Avena sativa L.: Conclusions on the conformation of the native protein. Planta 174, 396–401Google Scholar
  5. 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. Nucl. Acids Res. 13, 8543–8559Google Scholar
  6. Hunkapiller, M.W., Hewick, R.M., Dreyer, W.J., Hood, L.E. (1983a) High-sensitivity sequencing with a gas-phase sequenator. Methods Enzymol 91, 399–413Google Scholar
  7. Hunkapiller, M.W., Lujan, E., Ostrander, F., Hood, L.E. (1983b) Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol 91, 227–236Google Scholar
  8. Hunt, R.E., Pratt, L.H. (1979) Phytochrome immunoaffinity purification. Plant Physiol. 64, 332–336Google Scholar
  9. Jones, A.M., Quail, P.H. (1986) Quaternary structure of 124-kilodalton phytochrome from Avena sativa L. Biochemistry 25, 2987–2995Google Scholar
  10. Jones, A.M., Vierstra, R.D., Daniels, S.M., Quail, P.H. (1985) The role of separate molecular domains in the structure of phytochrome from etiolated Avena sativa L. Planta 164, 501–506Google Scholar
  11. Kelly, J.M., Lagarias, J.C. (1985) Photochemistry of 124-kilodalton Avena phytochrome under constant illumination in vitro. Biochemistry 24, 6003–6010Google Scholar
  12. Lagarias, J.C., Mercurio, F.M. (1985) Structure function studies on phytochrome. J. Biol. Chem. 260, 2415–2423Google Scholar
  13. Lagarias, J.C., Rapoport, H. (1980) Chromopeptides from phytochrome. The structure and linkage of the Pr form of the phytochrome chromophore. J. Am. Chem. Soc. 102, 4821–4828Google Scholar
  14. Marcus, F. (1985) Preferential cleavage at aspartyl-prolyl peptide bonds in dilute acid. Int. J. Prot. Res. 25, 542–546Google Scholar
  15. Merrill, C.R., Goldman, D., Van Keuren, M.L. (1983) Silver staining methods for polyacrylamide gel electrophoresis. Methods Enzymol 96, 230–239Google Scholar
  16. Quail, P.H., Barker, R.F., Colbert, J.T., Daniels, S.M., Hershey, H.P., Idler, K.B., Jones, A.M., Lissemore, J.L. (1986) Structural features of the phytochrome molecule and feedback regulation of the expression of its genes in Avena. In: Molecular biology of plant growth control, pp. 425–439. Fox, J.E., Jacobs, M., eds. Alan R. Liss, New YorkGoogle Scholar
  17. Quail, P.H., Gatz, C., Hershey, H.P., Jones, A.M., Lissemore, J.L., Parks, B.M., Sharrock, R.A., Barker, R.F., Idler, K., Murray, M.G., Koorneef, M., Kendrick, R.E. (1987) Molecular biology of phytochrome. In: Phytochrome and photoregulation of plants, pp. 23–37, M. Furuya, ed. Academic Press, Tokyo Orlando, Fld., USAGoogle Scholar
  18. Reiff, U., Eilfeld, P., Rüdiger, W. (1985) A photoreversible 39 kDalton fragment from the Pfr form of 124 kDalton oat phytochrome. Z. Naturforsch. 40c, 693–698Google Scholar
  19. Rüdiger, W., Thümmler, F., Cmiel, E., Schneider, S. (1983) Chromophore structure of the physiologically active form (Pfr) of phytochrome. Proc. Natl. Acad. Sci. USA 80, 6244–6248Google Scholar
  20. Russell, A.J., Fersht, A.R. (1986) Commerical samples of subtilisin BPN'. Nature 321, 733Google Scholar
  21. Sharrock, R.A., Lissemore, J.L., Quail, P.H. (1986) Nucleotide and amino acid sequence of a Cururbita phytochrome clone: Identification of conserved features by comparison with Avena phytochrome. Gene 47, 287–295Google Scholar
  22. Siegelman, H.W., Turner, B.C., Hendricks, S.B. (1966) The chromophore of phytochrome. Plant Physiol. 41, 1289–1292Google Scholar
  23. Smithes, O., Gibson, D., Fanning, E.M., Goodlfiesch, R.M., Gilman, J.G., Ballantyne, D.L. (1971) Quantitative procedures for use with the Edman-Begg sequenator. Partial sequences of two unusual immunoglobulin light chains. Biochemistry 10, 4912–4921Google Scholar
  24. Song, P.-S. (1985) The molecular model of phytochrome deduced from optical probes. In: Optical properties and structure of tetrapyrroles, pp. 311–348, Blauer, G., Sund, H., eds., Walter de Gruyter and Co., New YorkGoogle Scholar
  25. Thümmler, F., Rüdiger, W. (1984) Chromophore structure in phytochrome intermediates and bleached forms of phytochrome. Physiol. Plant. 60, 378–382Google Scholar
  26. Vierstra, R.D., Quail, P.H. (1982) Proteolysis alters the spectral properties of 124 kdalton phytochrome from Avena. Planta 156, 158–165Google Scholar
  27. Vierstra, R.D., Quail, P.H. (1983) Purification and initial characterization of 124-kilodalton phytochrome from Avena Biochemistry 22, 2498–2505Google Scholar
  28. Yamamoto, K.T., Furuya, M. (1983) Spectral properties of chromophore-containing fragments prepared from pea phytochrome by limited proteolysis. Plant Cell Physiol. 24, 713–718Google Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • Alan M. Jones
    • 1
  • Peter H. Quail
    • 2
  1. 1.Department of BiologyUniversity of North CarolinaChapel HillUSA
  2. 2.Department of BotanyUniversity of WisconsinMadisonUSA

Personalised recommendations