Assessing Protein Stability Under Different Light and Circadian Conditions

  • Takatoshi Kiba
  • Rossana HenriquesEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1398)


Plants use light as an indicator of time and space as well as the major energy source for photosynthesis. Due to the development of specific photoreceptors, plants can perceive a wide range of wavelengths and adjust their development accordingly to their surroundings. In addition to light, the circadian clock allows the anticipation of diurnal and seasonal changes thus providing organisms with the adequate physiological responses to ever changing surroundings, which are reflected in increased fitness and survival rate. Although initially described as a set of interconnected transcriptional loops, it is now accepted that posttranslational modifications are also important for proper clock function. In fact, not only the clock but also light signaling rely on posttranslational modifications, such as phosphorylation and ubiquitination, for proper signal transduction. We have designed a simple and yet reproducible method to determine protein stability and half-life under different light and circadian conditions. Our method only requires standard laboratory equipment, a relatively small amount of starting material and can be applied to young seedlings and mature plants. Besides our application to study light and circadian clock proteins, this protocol can be adapted to any other conditions that regulate protein stability.

Key words

Protein Degradation 26S proteasome Circadian clock Light signaling 


  1. 1.
    Kami C, Lorrain S, Hornitschek P, Fankhauser C (2010) Chapter Two – Light-regulated plant growth and development. In: Marja CPT (ed) Current topics in developmental biology, vol 91. Academic Press, pp 29–66. doi: 10.1016/S0070-2153(10)91002-8
  2. 2.
    Ito S, Song YH, Imaizumi T (2012) LOV domain-containing F-Box proteins: light-dependent protein degradation modules in Arabidopsis. Mol Plant 5(3):573–582. doi: 10.1093/mp/sss013 CrossRefPubMedGoogle Scholar
  3. 3.
    Dodd A, Salathia N, Hall A, Kevei E, Toth R, Nagy F, Hibberd J, Millar A, Webb A (2005) Plant circadian clocks increase photosynthesis, growth, survival, and competitive advantage. Science 309(5734):630–633CrossRefPubMedGoogle Scholar
  4. 4.
    Gendron JM, Pruneda-Paz JL, Doherty CJ, Gross AM, Kang SE, Kay SA (2012) Arabidopsis circadian clock protein, TOC1, is a DNA-binding transcription factor. Proc Natl Acad Sci U S A 109(8):3167–3172. doi: 10.1073/pnas.1200355109 PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Huang W, Pérez-García P, Pokhilko A, Millar AJ, Antoshechkin I, Riechmann JL, Mas P (2012) Mapping the core of the Arabidopsis circadian clock defines the network structure of the oscillator. Science 336(6077):75–79. doi: 10.1126/science.1219075 CrossRefPubMedGoogle Scholar
  6. 6.
    Pokhilko A, Fernandez AP, Edwards KD, Southern MM, Halliday KJ, Millar AJ (2012) The clock gene circuit in Arabidopsis includes a repressilator with additional feedback loops. Mol Syst Biol 8. doi:
  7. 7.
    Nakamichi N, Kiba T, Henriques R, Mizuno T, Chua N, Sakakibara H (2010) PSEUDO-RESPONSE REGULATORS 9, 7, and 5 are transcriptional repressors in the Arabidopsis circadian clock. Plant Cell 22(3):594–605PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Hemmes H, Henriques R, Jang I-C, Kim S, Chua N-H (2012) Circadian clock regulates dynamic chromatin modifications associated with Arabidopsis CCA1/LHY and TOC1 transcriptional rhythms. Plant Cell Physiol 53(12):2016–2029PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Henriques R, Mas P (2013) Chromatin remodeling and alternative splicing: Pre- and post-transcriptional regulation of the Arabidopsis circadian clock. Semin Cell Dev Biol 24(5):399–406, 10.1016/j.semcdb.2013.02.009 CrossRefPubMedGoogle Scholar
  10. 10.
    Malapeira J, Khaitova LC, Mas P (2012) Ordered changes in histone modifications at the core of the Arabidopsis circadian clock. Proc Natl Acad Sci U S A 109(52):21540–21545. doi: 10.1073/pnas.1217022110 PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Song YH, Ito S, Imaizumi T (2010) Similarities in the circadian clock and photoperiodism in plants. Curr Opin Plant Biol 13(5):594–603, doi: 10.1016/j.pbi.2010.05.004 PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Kusakina J, Dodd AN (2012) Phosphorylation in the plant circadian system. Trends Plant Sci 17(10):575–583, doi: 10.1016/j.tplants.2012.06.008 CrossRefPubMedGoogle Scholar
  13. 13.
    Kiba T, Henriques R, Sakakibara H, Chua N (2007) Targeted degradation of PSEUDO-RESPONSE REGULATOR5 by an SCFZTL complex regulates clock function and photomorphogenesis in Arabidopsis thaliana. Plant Cell 19(8):2516–2530PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Mas P, Kim W-Y, Somers D, Kay S (2003) Targeted degradation of TOC1 by ZTL modulates circadian function in Arabidopsis thaliana. Nature 426(6966):567–570CrossRefPubMedGoogle Scholar
  15. 15.
    Leivar P, Monte E (2014) PIFs: systems integrators in plant development. Plant Cell 26(1):56–78. doi: 10.1105/tpc.113.120857 PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Henriques R, Jang I-C, Chua N-H (2009) Regulated proteolysis in light-related signaling pathways. Curr Opin Plant Biol 12(1):49–56CrossRefPubMedGoogle Scholar
  17. 17.
    Lau OS, Deng XW (2012) The photomorphogenic repressors COP1 and DET1: 20 years later. Trends Plant Sci 17(10):584–593, doi: 10.1016/j.tplants.2012.05.004 CrossRefPubMedGoogle Scholar
  18. 18.
    Baudry A, Ito S, Song YH, Strait AA, Kiba T, Lu S, Henriques R, Pruneda-Paz JL, Chua N-H, Tobin EM, Kay SA, Imaizumi T (2010) F-Box proteins FKF1 and LKP2 act in concert with ZEITLUPE to control Arabidopsis clock progression. Plant Cell 22(3):606–622PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.RIKEN Center for Sustainable Resource ScienceYokohamaJapan
  2. 2.Center for Research in Agricultural Genomics (CRAG)Consortium CSIC-IRTA-UAB-UB, Parc de Recerca UAB, Edifici CRAG, Campus UABBarcelonaSpain

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