Phototropism pp 175-190 | Cite as

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

  • Koji Okajima
  • Satoru TokutomiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1924)


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.

Key words

Kinase assay LOV domain Photoreaction Phototropin Ser/Thr kinase Spectrophotometry 



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.


  1. 1.
    Briggs WR, Christie JM (2002) Phototropins 1 and 2: versatile plant blue-light receptors. Trends Plant Sci 7:204–210CrossRefGoogle Scholar
  2. 2.
    Liscum E, Briggs WR (1995) Mutations in the NPH1 locus of arabidopsis disrupt the perception of phototropic stimuli. Plant Cell 7:473–485CrossRefGoogle Scholar
  3. 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 291:2138–2141CrossRefGoogle Scholar
  4. 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 98:6969–6974CrossRefGoogle Scholar
  5. 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 414:656–660CrossRefGoogle Scholar
  6. 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 152:1391–1405CrossRefGoogle Scholar
  7. 7.
    Christie JM, Swartz TE, Bogomolni RA, Briggs WR (2002) Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function. Plant J 32:205–219CrossRefGoogle Scholar
  8. 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 276:36493–36500CrossRefGoogle Scholar
  9. 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 129:762–773CrossRefGoogle Scholar
  10. 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 69:1316–1321CrossRefGoogle Scholar
  11. 11.
    Harper SM, Neil LC, Gardner KH (2003) Structural basis of a phototropin light switch. Science 301:1541–1544CrossRefGoogle Scholar
  12. 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:e0124284CrossRefGoogle Scholar
  13. 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 590:139–147CrossRefGoogle Scholar
  14. 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 9:379–384CrossRefGoogle Scholar
  15. 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 287:40972–40981CrossRefGoogle Scholar
  16. 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 9:2331–2343CrossRefGoogle Scholar
  17. 17.
    Christie JM, Blackwood L, Petersen J, Sullivan S (2015) Plant flavoprotein photoreceptors. Plant Cell Physiol 56:401–413CrossRefGoogle Scholar
  18. 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 585:3391–3395CrossRefGoogle Scholar
  19. 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 126:3930–3931CrossRefGoogle Scholar
  20. 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 278:724–731CrossRefGoogle Scholar
  21. 21.
    Guo H, Kottke T, Hegemann P, Dick B (2005) The phot LOV2 domain and its interaction with LOV1. Biophys J 89:402–412CrossRefGoogle Scholar
  22. 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 114:9206–9211CrossRefGoogle Scholar
  23. 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 293:963–972CrossRefGoogle Scholar
  24. 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 289:413–422CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of Physics, Faculty of Science and TechnologyKeio UniversityHiyoshiJapan
  2. 2.Department of Biological Science, Graduate School of ScienceOsaka Prefecture UniversitySakaiJapan
  3. 3.Botanical GardensTohoku UniversitySendaiJapan

Personalised recommendations