Skip to main content
PMC home page
The Plant Cell logoLink to The Plant Cell
. 1993 May;5(5):565–575. doi: 10.1105/tpc.5.5.565

Carboxy-terminal deletion analysis of oat phytochrome A reveals the presence of separate domains required for structure and biological activity.

J R Cherry 1, D Hondred 1, J M Walker 1, J M Keller 1, H P Hershey 1, R D Vierstra 1
PMCID: PMC160294  PMID: 8518556

Abstract

A series of seven carboxy-terminal deletion mutants of oat phytochrome A were stably expressed in transgenic tobacco to localize phytochrome domains involved in chromophore attachment, spectral integrity, photoreversibility between the red light (Pr)- and far-red light (Pfr)-absorbing forms, dimerization, and biological activity. Amino acids necessary for chromophore attachment in vivo were localized to the amino-terminal 398 residues because mutant proteins this small had covalently bound chromophore. Deletion mutants from the carboxy terminus to residue 653 were spectrally indistinguishable from the full-length chromoprotein. In contrast, further truncation to residue 399 resulted in a chromoprotein with a bleached Pfr absorbance spectrum, Pr and Pfr absorbance maxima shifted toward shorter wavelengths, and reduced Pfr to Pr phototransformation efficiency. Thus, residues between 399 ad 652 are required for spectral integrity but are not essential for chromophore attachment. The sequence(s) between residues 919 and 1093 appears to be necessary for dimerization. Carboxy-terminal mutants containing this region behaved as dimers under nondenaturing conditions in vitro, whereas truncations without this region behaved as monomers. None of the plants expressing high levels of deletion mutants lacking the 35 carboxy-terminal amino acids displayed the light-exaggerated phenotype characteristic of plants expressing biologically active phytochrome A, even when the truncated phytochromes were expressed at levels 6- to 15-fold greater than that effective for the full-length chromoprotein. Collectively, these data show that the phytochrome protein contains several separable carboxy-terminal domains required for structure/function and identify a domain within 35 residues of the carboxy terminus that is critical for the biological activity of the photoreceptor in vivo.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Boylan M. T., Quail P. H. Oat Phytochrome Is Biologically Active in Transgenic Tomatoes. Plant Cell. 1989 Aug;1(8):765–773. doi: 10.1105/tpc.1.8.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boylan M. T., Quail P. H. Phytochrome a overexpression inhibits hypocotyl elongation in transgenic Arabidopsis. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10806–10810. doi: 10.1073/pnas.88.23.10806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Cherry J. R., Hondred D., Walker J. M., Vierstra R. D. Phytochrome requires the 6-kDa N-terminal domain for full biological activity. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5039–5043. doi: 10.1073/pnas.89.11.5039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Deforce L., Tomizawa K., Ito N., Farrens D., Song P. S., Furuya M. In vitro assembly of apophytochrome and apophytochrome deletion mutants expressed in yeast with phycocyanobilin. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10392–10396. doi: 10.1073/pnas.88.23.10392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Furuya M. Molecular properties and biogenesis of phytochrome I and II. Adv Biophys. 1989;25:133–167. doi: 10.1016/0065-227x(89)90006-3. [DOI] [PubMed] [Google Scholar]
  7. Hershey H. P., Barker R. F., Idler K. B., Lissemore J. L., Quail P. H. Analysis of cloned cDNA and genomic sequences for phytochrome: complete amino acid sequences for two gene products expressed in etiolated Avena. Nucleic Acids Res. 1985 Dec 9;13(23):8543–8559. doi: 10.1093/nar/13.23.8543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jabben M., Shanklin J., Vierstra R. D. Ubiquitin-phytochrome conjugates. Pool dynamics during in vivo phytochrome degradation. J Biol Chem. 1989 Mar 25;264(9):4998–5005. [PubMed] [Google Scholar]
  9. Jones A. M., Erickson H. P. Domain structure of phytochrome from Avena sativa visualized by electron microscopy. Photochem Photobiol. 1989 Apr;49(4):479–483. doi: 10.1111/j.1751-1097.1989.tb09198.x. [DOI] [PubMed] [Google Scholar]
  10. Kay S. A., Nagatani A., Keith B., Deak M., Furuya M., Chua N. H. Rice Phytochrome Is Biologically Active in Transgenic Tobacco. Plant Cell. 1989 Aug;1(8):775–782. doi: 10.1105/tpc.1.8.775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Keller J. M., Shanklin J., Vierstra R. D., Hershey H. P. Expression of a functional monocotyledonous phytochrome in transgenic tobacco. EMBO J. 1989 Apr;8(4):1005–1012. doi: 10.1002/j.1460-2075.1989.tb03467.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Lagarias J. C., Mercurio F. M. Structure function studies on phytochrome. Identification of light-induced conformational changes in 124-kDa Avena phytochrome in vitro. J Biol Chem. 1985 Feb 25;260(4):2415–2423. [PubMed] [Google Scholar]
  14. Nagatani A., Kay S. A., Deak M., Chua N. H., Furuya M. Rice type I phytochrome regulates hypocotyl elongation in transgenic tobacco seedlings. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5207–5211. doi: 10.1073/pnas.88.12.5207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Quail P. H. Phytochrome: a light-activated molecular switch that regulates plant gene expression. Annu Rev Genet. 1991;25:389–409. doi: 10.1146/annurev.ge.25.120191.002133. [DOI] [PubMed] [Google Scholar]
  16. Romanowski M., Song P. S. Structural domains of phytochrome deduced from homologies in amino acid sequences. J Protein Chem. 1992 Apr;11(2):139–155. doi: 10.1007/BF01025219. [DOI] [PubMed] [Google Scholar]
  17. Shanklin J., Jabben M., Vierstra R. D. Red light-induced formation of ubiquitin-phytochrome conjugates: Identification of possible intermediates of phytochrome degradation. Proc Natl Acad Sci U S A. 1987 Jan;84(2):359–363. doi: 10.1073/pnas.84.2.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Stockhaus J., Nagatani A., Halfter U., Kay S., Furuya M., Chua N. H. Serine-to-alanine substitutions at the amino-terminal region of phytochrome A result in an increase in biological activity. Genes Dev. 1992 Dec;6(12A):2364–2372. doi: 10.1101/gad.6.12a.2364. [DOI] [PubMed] [Google Scholar]
  19. Thümmler F., Dufner M., Kreisl P., Dittrich P. Molecular cloning of a novel phytochrome gene of the moss Ceratodon purpureus which encodes a putative light-regulated protein kinase. Plant Mol Biol. 1992 Dec;20(6):1003–1017. doi: 10.1007/BF00028888. [DOI] [PubMed] [Google Scholar]
  20. Vierstra R. D., Quail P. H. Spectral Characterization and Proteolytic Mapping of Native 120-Kilodalton Phytochrome from Cucurbita pepo L. Plant Physiol. 1985 Apr;77(4):990–998. doi: 10.1104/pp.77.4.990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wagner D., Tepperman J. M., Quail P. H. Overexpression of Phytochrome B Induces a Short Hypocotyl Phenotype in Transgenic Arabidopsis. Plant Cell. 1991 Dec;3(12):1275–1288. doi: 10.1105/tpc.3.12.1275. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Plant Cell are provided here courtesy of Oxford University Press

RESOURCES