Plant Molecular Biology

, Volume 63, Issue 5, pp 669–678 | Cite as

The serine-rich N-terminal region of Arabidopsis phytochrome A is required for protein stability

  • Santiago A. Trupkin
  • Dimitry Debrieux
  • Andreas Hiltbrunner
  • Christian Fankhauser
  • Jorge J. Casal


Deletion or substitution of the serine-rich N-terminal stretch of grass phytochrome A (phyA) has repeatedly been shown to yield a hyperactive photoreceptor when expressed under the control of a constitutive promoter in transgenic tobacco or Arabidopsis seedlings retaining their native phyA. These observations have lead to the proposal that the serine-rich region is involved in negative regulation of phyA signaling. To re-evaluate this conclusion in a more physiological context we produced transgenic Arabidopsis seedlings of the phyA-null background expressing Arabidopsis PHYA deleted in the sequence corresponding to amino acids 6–12, under the control of the native PHYA promoter. Compared to the transgenic seedlings expressing wild-type phyA, the seedlings bearing the mutated phyA showed normal responses to pulses of far-red (FR) light and impaired responses to continuous FR light. In yeast two-hybrid experiments, deleted phyA interacted normally with FHY1 and FHL, which are required for phyA accumulation in the nucleus. Immunoblot analysis showed reduced stability of deleted phyA under continuous red or FR light. The reduced physiological activity can therefore be accounted for by the enhanced destruction of the mutated phyA. These findings do not support the involvement of the serine-rich region in negative regulation but they are consistent with a recent report suggesting that phyA turnover is regulated by phosphorylation.


High-irradiance response Light signaling Phytochrome A Protein degradation Serine-rich domain 



Phytochrome A


Far-red light


Short days


Very-low-fluence response


High-irradiance response


Red-light-absorbing form of phytochrome


FR-absorbing form of phytochrome


Wild type


Full-length phyA



We thank Eberhard Schäfer (University of Freiburg) for allowing us to do the yeast two-hybrid experiments in his lab. This work was supported by the National Agency for the promotion of Science and Technology of Argentina (ANPCYT, grant BID 1728/OC-AR PICT 11631 to JJC), by University of Buenos Aires (grant G021 to JJC), by the Swiss National Science Foundation (grant PP00A−103005 to CF), the Human Frontier Science Program (HFSP) (grant RGY0016/2004-C to CF) and the University of Lausanne (to CF); by a fellowship from the HFSP to AH (LT00631/2003-C).

Supplementary material

11103_2006_9115_MOESM1_ESM.pdf (912 kb)
ESM 1 (PDF 911 kb)


  1. Barnes SA, Nishizawa NK, Quaggio RB, Whitelam GC, Chua N-H (1996) Far-red light blocks greening of Arabidopsis seedlings via a phytochrome A-mediated change in plastid development. Plant Cell 8:601–615PubMedCrossRefGoogle Scholar
  2. Botto JF, Sánchez RA, Whitelam GC, Casal JJ (1996) Phytochrome A mediates the promotion of seed germination by very low fluences of light and canopy shade light in Arabidopsis. Plant Physiol 110:439–444PubMedGoogle Scholar
  3. Boylan M, Douglas N, Quail PH (1994) Dominant negative suppression of Arabidopsis photoresponses by mutant phytochrome A sequences identifies spatially discrete regulatory domains in the photoreceptor. Plant Cell 6:449–460PubMedCrossRefGoogle Scholar
  4. Casal JJ, Yanovsky MJ, Luppi JP (2000) Two photobiological pathways of phytochrome A activity, only one of which shows dominant negative suppression by phytochrome B. Photochem Photobiol 71:481–486PubMedCrossRefGoogle Scholar
  5. Casal JJ, Davis SJ, Kirchenbauer DJ, Viczian A, Yanovsky MJ, Clough RC, Kircher S, Jordan-Beebe ET, Schäfer E, Nagy F, Vierstra RD (2002) The serine-rich N-terminal domain of oat phytochrome A helps regulate light responses and subnuclear localization of the photoreceptor. Plant Physiol 129:1127–1137PubMedCrossRefGoogle Scholar
  6. Cerdán PD, Staneloni RJ, Ortega J, Bunge MM, Rodriguez-Batiller J, Sánchez RA, Casal JJ (2000) Sustained but not transient phytochrome A signaling targets a region of a Lhcb1*2 promoter not necessary for phytochrome B action. Plant Cell 12:1203–1211PubMedCrossRefGoogle Scholar
  7. Chen M, Chory J, Fankhauser C (2004) Light signal transduction in higher plants. Annu Rev Genet 38:87–117PubMedCrossRefGoogle Scholar
  8. Cherry J, Hondred D, Walker J, Viestra R (1992) Phytochrome requires the 6-kDa N-terminal domain for full biological activity. Proc Natl Acad Sci USA 89:5039–5043PubMedCrossRefGoogle Scholar
  9. Clough RC, Vierstra RD (1997) Phytochrome degradation. Plant Cell Environ 20:713–721CrossRefGoogle Scholar
  10. Emmler K, Stockhaus J, Chua N-H, Schäfer E (1995) An amino-terminal deletion of rice phytochrome A results in a dominant negative suppression of tobacco phytochrome A activity in transgenic tobacco seedlings. Planta 197:103–110PubMedCrossRefGoogle Scholar
  11. Fankhauser C, Casal JJ (2004) Phenotypic characterization of a photomorphogenic mutant. Plant J 39:747–760PubMedCrossRefGoogle Scholar
  12. Furuya M, Song P-S (1994) Assembly and properties of holophytochrome. In: Kendrick RE, Kronenberg GHM (eds) Photomorphogenesis in plants, 2nd edn. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 105–140Google Scholar
  13. Hennig L, Büche C, Eichenberg K, Schäfer E (1999) Dynamic properties of endogenous phytochrome A in Arabidopsis seedlings. Plant Physiol 121:571–578PubMedCrossRefGoogle Scholar
  14. Hennig L, Büche C, Schäfer E (2000) Degradation of phytochrome A and the high irradiance response of Arabidopsis: a kinetic analysis. Plant Cell Environ 23:727–734CrossRefGoogle Scholar
  15. Hiltbrunner A, Viczian A, Bury E, Tscheuschler A, Kircher S, Tóth R, Honsberger A, Nagy F, Fankhauser C, Schäfer E (2005) Nuclear accumulation of the phytochrome A photoreceptor requires FHY1. Curr Biol 15:2125–2130PubMedCrossRefGoogle Scholar
  16. Hiltbrunner A, Tscheuschler A, Viczian A, Kunkel T, Kircher S, Schäfer E (2006) FHY1 and FHL act together to mediate nuclear accumulation of the phytochrome A photoreceptor. Plant Cell Physiol 47:1023–1034PubMedCrossRefGoogle Scholar
  17. Hisada A, Hanzawa H, Weller JL, Nagatani A, Reid JB, Furuya M (2000) Light-induced nuclear translocation of endogenous pea phytochrome A visualized by immunocytochemical procedures. Plant Cell 12:1063–1078PubMedCrossRefGoogle Scholar
  18. Johnson E, Bradley M, Harberd P, Whitelam GC (1994) Photoresponses of light-grown phyA mutants of Arabidopsis. Phytochrome A is required for the perception of daylength extensions. Plant Physiol 105:141–149PubMedCrossRefGoogle Scholar
  19. Jordan ET, Cherry JR, Walker JM, Vierstra RD (1995) The amino-terminus of phytochrome A contains two distinct functional domains. Plant J 9:243–257CrossRefGoogle Scholar
  20. Jordan ET, Marita JM, Clough RC, Vierstra RD (1997) Characterization of regions within the N-terminal 6-kilodalton domain of phytochrome A that modulate its biological activity. Plant Physiol 115:693–704PubMedCrossRefGoogle Scholar
  21. Kim J-I, Shen Y, Han Y-J, Park J-E, Kirchenbauer D, Soh M-S, Nagy F, Schäfer E, Song P-S (2004) Phytochrome phosphorylation modulates light signaling by influencing the protein–protein interaction. Plant Cell 16:2629–2640PubMedCrossRefGoogle Scholar
  22. Kircher S, Kozma-Bognar L, Kim L, Adam E, Harter K, Schäfer E, Nagy F (1999) Light-quality dependent nuclear import of the plant photoreceptors phytochrome A and B. Plant Cell 11:1445–1456PubMedCrossRefGoogle Scholar
  23. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  24. Lapko VN, Jiang X-Y, Smith DL, Song P-S (1997) Posttranslational modification of oat phytochrome A: phosphorylation of a specific serine in a multiple serine cluster. Biochemistry 36:10595–10599PubMedCrossRefGoogle Scholar
  25. Lapko VN, Jiang XY, Smith DL, Song PS (1999) Mass spectrometric characterization of oat phytochrome A: isoforms and posttranslational modifications. Protein Sci 8:1032–1044. Google Scholar
  26. Mancinelli AL, Rossi F, Moroni A (1991) Cryptochrome, phytochrome and anthocyanin production. Plant Physiol 96:1079–1085PubMedGoogle Scholar
  27. Moran R (1982) Formulae for determination of chlorophyllous pigments extracted with N,N-dimethylformamide. Plant Physiol 69:1376–1381PubMedCrossRefGoogle Scholar
  28. Quail PH, Boylan MT, Parks BM, Short TW, Xu Y, Wagner D (1995) Phytochromes: photosensory perception and signal transduction. Science 268:675–680PubMedCrossRefGoogle Scholar
  29. Ryu JS, Kim JI, Kunkel T, Kim BC, Cho DS, Hong SH, Kim SH, Fernandez AP, Kim Y, Alonso JM, Ecker JR, Nagy F, Lim PO, Song PS, Schäfer E, Nam HG (2005) Phytochrome-specific type 5 phosphatase controls light signal flux by enhancing phytochrome stability and affinity for a signal transducer. Cell 120:395–406PubMedCrossRefGoogle Scholar
  30. Schäfer E, Nagy F (2006) Photomorphogenesis in plants and bacteria: function and signal transduction mechanisms. Springer, Berlin Heidelberg, New YorkCrossRefGoogle Scholar
  31. Schmidt TGM, Koepke J, Frank R, Skerra A (1996) Molecular interactions between the Strep-tag affinity peptide and its cognate target, streptavidin. J Mol Biol 255:753–766PubMedCrossRefGoogle Scholar
  32. Schumacher K, Vafeados D, McCarthy M, Sze H, Wilkins T, Chory J (1999) The Arabidopsis det3 mutant reveals a central role for the vacuolar H+-ATPase in plant growth and development. Genes Dev 13:3259–3270PubMedCrossRefGoogle Scholar
  33. Seo HS, Watanabe E, Tokutomi S, Nagatani A, Chua N-H (2004) Photoreceptor ubiquitination by COP1 E3 ligase desensitizes phytochrome A signaling. Genes Dev 18:617–622PubMedCrossRefGoogle Scholar
  34. Shimizu-Sato S, Huq E, Tepperman JM, Quail PH (2002) A light-switchable gene promoter system. Nat Biotechnol 20:1041–1044PubMedCrossRefGoogle Scholar
  35. Shinomura T, Nagatani A, Hanzawa H, Kubota M, Watanabe M, Furuya M (1996) Action spectrum for phytochrome-A and B-specific photoinduction of seed germination in Arabidopsis thaliana. Proc Natl Acad Sci USA 93:8129–8133PubMedCrossRefGoogle Scholar
  36. Shinomura T, Uchida K, Furuya M (2000) Elementary processes of photoperception by phytochrome A for high irradiance response of hypocotyl elongation in Arabidopsis thaliana. Plant Physiol 122:147–156PubMedCrossRefGoogle Scholar
  37. Stockhaus J, Nagatani A, Halfter U, Kay S, Furuya M, Chua N-H (1992) Serine-to-alanine substitutions at the amino-terminal region of phytochrome A result in an increase in biological activity. Genes Dev 6:2364–2372PubMedGoogle Scholar
  38. Tepperman JM, Zhu T, Chang H-S, Wang X, Quail PH (2001) Multiple transcription-factor genes are early targets of phytochrome A signaling. Proc Natl Acad Sci USA 98:9437–9442PubMedCrossRefGoogle Scholar
  39. Wagner JR, Brunzelle JS, Forest KT, Vierstra RD (2005) A light-sensing knot revealed by the structure of the chromophore-binding domain of phytochrome. Nature 438:325–331PubMedCrossRefGoogle Scholar
  40. Whitelam GC, Johnson E, Peng J, Carol P, Anderson ML, Cowl JS, Harberd NP (1993) Phytochrome A null mutants of Arabidopsis display a wild-type phenotype in white light. Plant Cell 5:757–768PubMedCrossRefGoogle Scholar
  41. Yanovsky MJ, Casal JJ, Luppi JP (1997) The VLF loci, polymorphic between ecotypes Landsberg erecta and Columbia dissect two branches of phytochrome A signalling pathways that correspond to the very-low fluence and high-irradiance responses of phytochrome. Plant J 12:659–667PubMedCrossRefGoogle Scholar
  42. Yanovsky MJ, Whitelam GC, Casal JJ (2000) fhy3-1 retains inductive responses of phytochrome A. Plant Physiol 123:235–242PubMedCrossRefGoogle Scholar
  43. Yanovsky MJ, Luppi JP, Kirchbauer D, Ogorodnikova OB, Sineshchekov VA, Adam E, Kircher S, Staneloni RJ, Schäfer E, Nagy F, Casal JJ (2002) Missense mutation in the PAS2 domain of phytochrome A impairs subnuclear localization and a subset of responses. Plant Cell 14:1591–1603PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Santiago A. Trupkin
    • 1
  • Dimitry Debrieux
    • 2
  • Andreas Hiltbrunner
    • 3
  • Christian Fankhauser
    • 2
  • Jorge J. Casal
    • 1
  1. 1.IFEVA, Facultad de AgronomíaUniversidad de Buenos Aires and CONICETBuenos AiresArgentina
  2. 2.Center for Integrative GenomicsUniversity of LausanneLausanneSwitzerland
  3. 3.Institute of Biology II/BotanyUniversity of FreiburgFreiburgGermany

Personalised recommendations