Skip to main content
SpringerLink
Log in
SpringerLink
Log in

Measurements of Photoreaction and Kinase Activity of Phototropin, a Photoreceptor Protein for Tropic Response in Plants: Involvement of Kinase Activity in the Photosensitivity of Tropic Response

  • Protocol
  • First Online:
Phototropism

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1924))

  • 781 Accesses

Abstract

Phototropin is a photoreceptor protein responsible for phototropic responses in plants. A phototropin molecule has two photoreceptive domains named LOV1 and LOV2 in the N-terminal region. Blue light absorbed by a chromophore in these domains triggers conformational changes in the protein moiety. The C-terminal region of phototropin forms a Ser/Thr kinase that is activated by these conformational changes. The activated phototropin kinase transmits signals downstream leading to tropic responses. The lifetime of the activated state may concern the sensitivity of the tropic responses to light. Thus, spectrophotometric and kinase activity analyses of phototropin are important to understand the light signaling processes related to the photosensitivity. The preparation of polypeptide samples of Arabidopsis phototropin and the methods of spectroscopic measurements and kinase assay of these samples are shown in this chapter.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Briggs WR, Christie JM (2002) Phototropins 1 and 2: versatile plant blue-light receptors. Trends Plant Sci 7:204–210

    Article  CAS  Google Scholar 

  2. Liscum E, Briggs WR (1995) Mutations in the NPH1 locus of arabidopsis disrupt the perception of phototropic stimuli. Plant Cell 7:473–485

    Article  CAS  Google Scholar 

  3. Kagawa T, Sakai T, Suetsugu N, Oikawa K, Ishiguro S, Kato T, Tabata S, Okada K, Wada M (2001) Arabidopsis NPL1: a phototropin homolog controlling the chloroplast high-light avoidance response. Science 291:2138–2141

    Article  CAS  Google Scholar 

  4. Sakai T, Kagawa T, Kasahara M, Swartz T, Christie JM, Briggs WR, Wada M, Okada K (2001) Arabidopsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation. Proc Natl Acad Sci USA 98:6969–6974

    Article  CAS  Google Scholar 

  5. Kinoshita T, Doi M, Suetsugu N, Kagawa T, Wada M, Shimazaki K (2001) phot1 and phot2 mediate blue light regulation of stomatal opening. Nature 414:656–660

    Article  CAS  Google Scholar 

  6. de Carbonnel M, Davis P, Roelfsema MRG, Inoue S, Schepens I, Lariguet P, Geisler M, Shimazaki K, Hangarter R, Fankhauser C (2010) The Arabidopsis PHYTOCHROME KINASE SUBSTRATE2 protein is a phototropin signaling element that regulates leaf flattening and leaf positioning. Plant Physiol 152:1391–1405

    Article  Google Scholar 

  7. Christie JM, Swartz TE, Bogomolni RA, Briggs WR (2002) Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function. Plant J 32:205–219

    Article  CAS  Google Scholar 

  8. Swartz TE, Corchnoy SB, Christie JM, Lewis JW, Szundi I, Briggs WR, Bogomolni RA (2001) The photocycle of a flavin-binding domain of the blue light photoreceptor phototropin. J Biol Chem 276:36493–36500

    Article  CAS  Google Scholar 

  9. Kasahara M, Swartz TE, Olney MA, Onodera A, Mochizuki N, Fukuzawa F, Asamizu E, Tabata S, Kanegae N, Takano M, Christie JM, Nagatani A, Briggs WR (2002) Photochemical properties of the favin mononucleotide-binding domains of the phototropins from Arabidopsis, rice, and Chlamydomonas reinhardtii. Plant Physiol 129:762–773

    Article  CAS  Google Scholar 

  10. Halavaty AS, Moffat K (2013) Coiled-coil dimerization of the LOV2 domain of the blue-light photoreceptor phototropin 1 from Arabidopsis thaliana. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:1316–1321

    Article  CAS  Google Scholar 

  11. Harper SM, Neil LC, Gardner KH (2003) Structural basis of a phototropin light switch. Science 301:1541–1544

    Article  CAS  Google Scholar 

  12. Kashojiya S, Okajima K, Shimada T, Tokutomi S (2015) Essential role of the A’α/Aβ gap in the N-terminal upstream of LOV2 for the blue light signaling from LOV2 to kinase in Arabidopsis photototropin1, a plant blue light receptor. PLoS One 10:e0124284

    Article  Google Scholar 

  13. Kashojiya S, Yoshihara S, Okajima K, Tokutomi S (2016) The linker between LOV2-Jα and STK plays an essential role in the kinase activation by blue light in Arabidopsis phototropin1, a plant blue light receptor. FEBS Lett 590:139–147

    Article  CAS  Google Scholar 

  14. Strickland D, Lin Y, Wagner EC, Hope CM, Zayner J, Antoniou C, Sosnick TR, Weiss EL, Glotze M (2012) TULIPs: tunable, light-controlled interacting protein tags for cell biology. Nat Methods 9:379–384

    Article  CAS  Google Scholar 

  15. Okajima K, Kashojiya S, Tokutomi S (2012) Photosensitivity of kinase activation by blue light involves the lifetime of a cysteinyl-flavin adduct intermediate, S390, in the photoreaction cycle of the LOV2 domain in phototropin, a plant blue light receptor. J Biol Chem 287:40972–40981

    Article  CAS  Google Scholar 

  16. van der Steen JB, Nakasone Y, Hendriks J, Hellingwerf KJ (2013) Modeling the functioning of YtvA in the general stress response in Bacillus subtilis. Mol BioSyst 9:2331–2343

    Article  Google Scholar 

  17. Christie JM, Blackwood L, Petersen J, Sullivan S (2015) Plant flavoprotein photoreceptors. Plant Cell Physiol 56:401–413

    Article  CAS  Google Scholar 

  18. Okajima K, Matsuoka D, Tokutomi S (2011) LOV2-linker-kinase phosphorylates LOV1-containing N-terminal polypeptide substrate via photoreaction of LOV2 in Arabidopsis phototropin1. FEBS Lett 585:3391–3395

    Article  CAS  Google Scholar 

  19. Harper SM, Neil LC, Day IJ, Hore PJ, Gardner KH (2004) Conformational changes in a photosensory LOV domain monitored by time-resolved NMR spectroscopy. J Am Chem Soc 126:3930–3931

    Article  Google Scholar 

  20. Corchnoy SB, Swartz TE, Lewis JW, Szundi I, Briggs WR, Bogomolni RA (2003) Intramolecular proton transfers and structural changes during the photocycle of the LOV2 domain of phototropin 1. J Biol Chem 278:724–731

    Article  CAS  Google Scholar 

  21. Guo H, Kottke T, Hegemann P, Dick B (2005) The phot LOV2 domain and its interaction with LOV1. Biophys J 89:402–412

    Article  CAS  Google Scholar 

  22. Fujii Y, Tanaka H, Konno K, Ogasawara Y, Hamashima N, Tamura S, Hasegawa S, Hayasaki Y, Okajima K, Kodama Y (2017) Phototropin perceives temperature based on the lifetime of its photoactivated state. Proc Natl Acad Sci USA 114:9206–9211

    Article  CAS  Google Scholar 

  23. Oide M, Okajima K, Nakagami H, Kato T, Sekiguchi Y, Oroguchi T, Hikima T, Yamamoto T, Nakasako M (2018) Blue light-excited LOV1 and LOV2 domains cooperatively regulate the kinase activity of full-length phototropin2 from Arabidopsis. J Biol Chem 293:963–972

    Article  CAS  Google Scholar 

  24. Okajima K, Aihara Y, Takayama Y, Nakajima Y, Kashojiya S, Hikima T, Oroguchi T, Kobayashi A, Sekiguchi Y, Yamamoto M, Suzuki S, Nagatani A, Nakasako M, Tokutomi S (2014) Light-induced conformational changes of LOV1 (light oxygen voltage-sensing domain 1) and LOV2 relative to the kinase domain and regulation of kinase activity in Chlamydomonas Phototropin. J Biol Chem 289:413–422

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported in part by Grant-in-Aid for Scientific Research on Innovative Areas to S.T. (22120005) and Grant-in-Aid for Scientific Research on Priority Areas to S.T. (17084008) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and Grant-in-Aid for Exploratory Research to S.T. (23657105) and Grant-in-aid for Young Scientist B to K.O. (15K18559) from the Japan Society for the Promotion of Science.

Author information

Authors and Affiliations

  1. Department of Physics, Faculty of Science and Technology, Keio University, Hiyoshi, Yokohama, Japan

    Koji Okajima

  2. Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka, Japan

    Koji Okajima & Satoru Tokutomi

  3. Botanical Gardens, Tohoku University, Sendai, Japan

    Satoru Tokutomi

Authors
  1. Koji Okajima
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Satoru Tokutomi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Satoru Tokutomi .

Editor information

Editors and Affiliations

  1. Department of Biological Sciences, Faculty of Science, Hokkaido University, Sapporo, Japan

    Kotaro T. Yamamoto

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Okajima, K., Tokutomi, S. (2019). Measurements of Photoreaction and Kinase Activity of Phototropin, a Photoreceptor Protein for Tropic Response in Plants: Involvement of Kinase Activity in the Photosensitivity of Tropic Response. In: Yamamoto, K. (eds) Phototropism. Methods in Molecular Biology, vol 1924. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9015-3_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9015-3_14

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9014-6

  • Online ISBN: 978-1-4939-9015-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation