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. 1997 Dec;9(12):2271–2280. doi: 10.1105/tpc.9.12.2271

Biochemical characterization of Arabidopsis wild-type and mutant phytochrome B holoproteins.

T D Elich 1, J Chory 1
PMCID: PMC157073  PMID: 9437866

Abstract

Although phytochrome B (phyB) plays a particularly important role throughout the life cycle of a plant, it has not been studied in detail at the molecular level due to its low abundance. Here, we report on the expression, assembly with chromophore, and purification of epitope-tagged Arabidopsis phyB. In addition, we have reconstructed two missense mutations, phyB-4 and phyB-101, isolated in long hypocotyl screens. We show that mutant proteins phyB-4 and phyB-101 exhibit altered spectrophotometric and biochemical properties relative to the wild-type protein. In particular, we demonstrate that phyB-101 Pfr exhibits rapid nonphotochemical (dark) reversion to Pr that results in a lower photoequilibrium level of the active Pfr form. We conclude that this occurs in vivo as well because phyB-101 mutants are shown to lack an end-of-day-far-red hypocotyl elongation response that requires a stable Pfr species. We propose that this Pfr instability may be the primary molecular mechanism underlying the phyB-101 mutant phenotype.

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Selected References

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  1. Andel F., 3rd, Lagarias J. C., Mathies R. A. Resonance raman analysis of chromophore structure in the lumi-R photoproduct of phytochrome. Biochemistry. 1996 Dec 17;35(50):15997–16008. doi: 10.1021/bi962175k. [DOI] [PubMed] [Google Scholar]
  2. Casal J. J. Phytochrome A enhances the promotion of hypocotyl growth caused by reductions in levels of phytochrome B in its far-red-light-absorbing form in light-grown Arabidopsis thaliana. Plant Physiol. 1996 Nov;112(3):965–973. doi: 10.1104/pp.112.3.965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cherry J. R., Hondred D., Walker J. M., Keller J. M., Hershey H. P., Vierstra R. D. Carboxy-terminal deletion analysis of oat phytochrome A reveals the presence of separate domains required for structure and biological activity. Plant Cell. 1993 May;5(5):565–575. doi: 10.1105/tpc.5.5.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Elich T. D., Chory J. Initial events in phytochrome signalling: still in the dark. Plant Mol Biol. 1994 Dec;26(5):1315–1327. doi: 10.1007/BF00016477. [DOI] [PubMed] [Google Scholar]
  5. Elich T. D., Lagarias J. C. Formation of a photoreversible phycocyanobilin-apophytochrome adduct in vitro. J Biol Chem. 1989 Aug 5;264(22):12902–12908. [PubMed] [Google Scholar]
  6. Hamazato F., Shinomura T., Hanzawa H., Chory J., Furuya M. Fluence and wavelength requirements for Arabidopsis CAB gene induction by different phytochromes. Plant Physiol. 1997 Dec;115(4):1533–1540. doi: 10.1104/pp.115.4.1533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kay S. A. PAS, present, and future: clues to the origins of circadian clocks. Science. 1997 May 2;276(5313):753–754. doi: 10.1126/science.276.5313.753. [DOI] [PubMed] [Google Scholar]
  8. Kieber J. J., Rothenberg M., Roman G., Feldmann K. A., Ecker J. R. CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the raf family of protein kinases. Cell. 1993 Feb 12;72(3):427–441. doi: 10.1016/0092-8674(93)90119-b. [DOI] [PubMed] [Google Scholar]
  9. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  10. Kunkel T., Speth V., Büche C., Schäfer E. In vivo characterization of phytochrome-phycocyanobilin adducts in yeast. J Biol Chem. 1995 Aug 25;270(34):20193–20200. doi: 10.1074/jbc.270.34.20193. [DOI] [PubMed] [Google Scholar]
  11. Kunkel T., Tomizawa K., Kern R., Furuya M., Chua N. H., Schäfer E. In vitro formation of a photoreversible adduct of phycocyanobilin and tobacco apophytochrome B. Eur J Biochem. 1993 Aug 1;215(3):587–594. doi: 10.1111/j.1432-1033.1993.tb18069.x. [DOI] [PubMed] [Google Scholar]
  12. Lagarias D. M., Wu S. H., Lagarias J. C. Atypical phytochrome gene structure in the green alga Mesotaenium caldariorum. Plant Mol Biol. 1995 Dec;29(6):1127–1142. doi: 10.1007/BF00020457. [DOI] [PubMed] [Google Scholar]
  13. Li L., Lagarias J. C. Phytochrome assembly. Defining chromophore structural requirements for covalent attachment and photoreversibility. J Biol Chem. 1992 Sep 25;267(27):19204–19210. [PubMed] [Google Scholar]
  14. Murphy J. T., Lagarias J. C. Purification and characterization of recombinant affinity peptide-tagged oat phytochrome A. Photochem Photobiol. 1997 Apr;65(4):750–758. doi: 10.1111/j.1751-1097.1997.tb01920.x. [DOI] [PubMed] [Google Scholar]
  15. Nagatani A., Reed J. W., Chory J. Isolation and Initial Characterization of Arabidopsis Mutants That Are Deficient in Phytochrome A. Plant Physiol. 1993 May;102(1):269–277. doi: 10.1104/pp.102.1.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Parks B. M., Quail P. H. hy8, a new class of arabidopsis long hypocotyl mutants deficient in functional phytochrome A. Plant Cell. 1993 Jan;5(1):39–48. doi: 10.1105/tpc.5.1.39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Quail P. H., Boylan M. T., Parks B. M., Short T. W., Xu Y., Wagner D. Phytochromes: photosensory perception and signal transduction. Science. 1995 May 5;268(5211):675–680. doi: 10.1126/science.7732376. [DOI] [PubMed] [Google Scholar]
  18. Reed J. W., Nagpal P., Poole D. S., Furuya M., Chory J. Mutations in the gene for the red/far-red light receptor phytochrome B alter cell elongation and physiological responses throughout Arabidopsis development. Plant Cell. 1993 Feb;5(2):147–157. doi: 10.1105/tpc.5.2.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ruddat A., Schmidt P., Gatz C., Braslavsky S. E., Gärtner W., Schaffner K. Recombinant type A and B phytochromes from potato. Transient absorption spectroscopy. Biochemistry. 1997 Jan 7;36(1):103–111. doi: 10.1021/bi962012w. [DOI] [PubMed] [Google Scholar]
  20. Shinomura T., Nagatani A., Hanzawa H., Kubota M., Watanabe M., Furuya M. Action spectra for phytochrome A- and B-specific photoinduction of seed germination in Arabidopsis thaliana. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):8129–8133. doi: 10.1073/pnas.93.15.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Terry M. J., Maines M. D., Lagarias J. C. Inactivation of phytochrome- and phycobiliprotein-chromophore precursors by rat liver biliverdin reductase. J Biol Chem. 1993 Dec 15;268(35):26099–26106. [PubMed] [Google Scholar]
  22. Wagner D., Quail P. H. Mutational analysis of phytochrome B identifies a small COOH-terminal-domain region critical for regulatory activity. Proc Natl Acad Sci U S A. 1995 Sep 12;92(19):8596–8600. doi: 10.1073/pnas.92.19.8596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Wahleithner J. A., Li L. M., Lagarias J. C. Expression and assembly of spectrally active recombinant holophytochrome. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10387–10391. doi: 10.1073/pnas.88.23.10387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Whitelam G. C., Johnson E., Peng J., Carol P., Anderson M. L., Cowl J. S., Harberd N. P. Phytochrome A null mutants of Arabidopsis display a wild-type phenotype in white light. Plant Cell. 1993 Jul;5(7):757–768. doi: 10.1105/tpc.5.7.757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yeh K. C., Wu S. H., Murphy J. T., Lagarias J. C. A cyanobacterial phytochrome two-component light sensory system. Science. 1997 Sep 5;277(5331):1505–1508. doi: 10.1126/science.277.5331.1505. [DOI] [PubMed] [Google Scholar]

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