Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- Da:
-
Dalton
- HA:
-
hydroxyapatite
- MAb:
-
monoclonal antibody
- PAb:
-
polyclonal antibody preparation
- PAGE:
-
polyacrylamide gel electrophoresis
- SDS:
-
sodium dodecyl sulfate
References
Abe, H., Yamamoto, K.T., Nagatani, A., Furuya, M. (1985) Characterization of green tissue-specific phytochrome isolated immunochemically from pea seedlings. Plant Cell Physiol. 26, 1387–1399
Berkelman, T., Lagarias, J.C. (1986) Visualization of bilin-linked peptides and proteins in polyacrylamide gels. Anal. Biochem. 156, 194–201
Cordonnier, M-M., Smith, C., Greppin, H., Pratt, L.H. (1983) Production and purification of monoclonal antibodies to Pisum and Avena phytochrome. Planta 158, 369–376
Cordonnier, M.-M., Greppin, H., Pratt, L.H. (1985) Monoclonal antibodies with differing affinities to the red- and far-red-absorbing forms of phytochrome. Biochemistry 24, 3246–3253
Cordonnier, M.-M., Greppin, H., Pratt, L.H. (1986a) Identification of a highly conserved domain on phytochrome from angiosperms to algae. Plant Physiol. 80, 982–987
Cordonnier, M.-M., Greppin H., Pratt, L.H. (1986b) Phytochrome from green Avena shoots characterized with a monoclonal antibody to phytochrome from etiolated Pisum shoots. Biochemistry 25, 7657–7666
Glover, C.V.C. III (1989) Sequence-specific protein-DNA recognition by transcriptional regulatory proteins. Plant Mol. Biol. Reporter 7, 183–208
Jones, A.M., Quail, P.H. (1986) Quaternary structure of 124-kilodalton phytochrome from Avena sativa L. Biochemistry 25, 2987–2995
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685
Mierendorf, R.C., Percy, C., Young, R.A. (1987) Gene isolation by screening γgt11 libraries with antibodies. Methods Enzymol. 152, 458–469
Pratt, L.H. (1984) Phytochrome purification. In: Techniques in photomorphogenesis, pp. 175–200, Smith, H., Holmes, M.G., eds. Academic Press, London New York
Pratt, L.H., McCurdy, D.W., Shimazaki, Y., Cordonnier, M.-M. (1986) Immunodetection of phytochrome: immunocytochemistry, immunoblotting, and immunoquantitation. In: Modern methods of plant analysis, N.S., vol 4, pp. 50–74, Linskens, H.F., Jackson, J.F., eds. Springer, Berlin Heidelberg New York
Pratt, L.H., Shimazaki, Y., Stewart, S.J., Cordonnier, M.-M. (1991a) Large-scale partial purification of phytochrome from green leaves of Avena sativa L. Planta 184, 81–86
Pratt, L.H., Stewart, S.J., Shimazaki, Y., Wang, Y.-C., Cordonnier, M.-M. (1991b) Monoclonal antibodies directed to phytochrome from green leaves of Avena sativa L. cross-react weakly or not at all with the phytochrome that is most abundant in etiolated shoots of the same species. Planta 184, 87–95
Pratt, L.H., Cordonnier, M.-M., Wang, Y.-C., Stewart, S.J., and Moyer, M. (1991c) Evidence for three phytochromes in Avena. In: Properties and biological action of phytochrome, Thomas, B., ed. Springer, Berlin, in press
Sharrock, R.A., Quail, P.H. (1989) Novel phytochrome sequences in Arabidopsis thaliana: structure, evolution, and differential expression of a plant regulatory gene family. Genes Dev. 3, 1745–1757
Shimazaki, Y., Pratt, L.H. (1985) Immunochemical detection with rabbit polyclonal and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green Avena shoots. Planta 164, 333–344
Shimazaki, Y., Pratt, L.H. (1986) Immunoprecipitation of phytochrome from green Avena by rabbit antisera to phytochrome from etiolated Avena. Planta 168, 512–515
Shimazaki, Y., Cordonnier, M.-M., Pratt, L.H. (1983) Phytochrome quantitation in crude extracts of Avena by enzymelinked immunosorbent assay with monoclonal antibodies. Planta 159, 534–544
Shimazaki, Y., Cordonnier, M.-M., Pratt, L.H. (1986) Identification with monoclonal antibodies of a second antigenic domain on Avena phytochrome that changes upon its photoconversion. Plant Physiol. 82, 109–113
Sommer, D., Song, P.-S. (1990) Chromophore topography and secondary structure of 124-kilodalton Avena phytochrome probed by zinc-induced chromophore modification. Biochemistry 29, 1943–1948
Thompson, L.K., Pratt, L.H., Cordonnier, M.-M., Kadwell, S., Darlix, J.-L., Crossland, L. (1989) Fusion protein-based epitope mapping of phytochrome: precise identification of an evolutionarily conserved domain. J. Biol. Chem. 264, 12426–12431
Tokuhisa, J.G., Quail, P.H. (1989) Phytochrome in green tissue: partial purification and characterization of the 118-kilodalton phytochrome species from light-grown Avena sativa L. Photochem. Photobiol. 50, 143–152
Tokuhisa, J.G., Daniels, S.M., Quail, P.H. (1985) 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
Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354
Vierstra, R.D., Quail, P.H. (1983) Purification and initial characterization of 124-kilodalton phytochrome from Avena. Biochemistry 22, 2498–2505
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
Author information
Authors and Affiliations
Additional information
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.
Rights and permissions
About this article
Cite this article
Wang, YC., Stewart, S.J., Cordonnier, MM. et al. Avena sativa L. contains three phytochromes, only one of which is abundant in etiolated tissue. Planta 184, 96–104 (1991). https://doi.org/10.1007/BF00208242
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00208242