Skip to main content
SpringerLink
Log in

Differential Protein Phosphorylation Regulates Chloroplast Movement in Response to Strong Light and Darkness in Arabidopsis thaliana

  • Original Paper
  • Published:
Plant Molecular Biology Reporter Aims and scope Submit manuscript

Abstract

Phototropin-dependent chloroplast movement is essential to the photosynthetic acclimation of mesophyll cells to incident light. Chloroplast movement involves many cellular actors, such as chloroplast-associated actin filaments and proteins that mediate signalling between phototropins and chloroplast motion. In the past few years, genetic approaches have identified several key proteins but the intrinsic mechanisms of the signalling cascade, such as phosphorylation events, remain undefined. Here, we took advantage of phosphoproteomics to examine the involvement of protein phosphorylation in chloroplast movement in darkness or under high light, at different CO2 mole fractions (100, 380 or 1,000 ppm) to vary photosynthetic activity. Amongst the 100 relevant identified phosphopeptides, 19 (corresponding to 8 proteins) were differentially phosphorylated in darkness vs. high light. There was no significant CO2 effect on the observed phosphorylation patterns. We further characterized the phosphorylation sites in THRUMIN1, which is believed to be crucial for the attachment of chloroplast-associated actin filaments to the plasma membrane and thus for chloroplast movements. The mutant thrumin1 was complemented with a mutated protein in which phospho-sites were substituted to a phosphomimetic (Asp) or a non-phosphorylatable (Ala) residue. While the phosphomimetic substitution altered the chloroplast response in the light only, both light and dark responses were altered with the non-phosphorylatable substitution. Our data suggest a key role of protein phosphorylation, including that of THRUMIN1, in the light/dark control of chloroplast movements.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

BL:

blue light

cp-actin:

chloroplast-associated actin

phot:

phototropin

References

  • Aggarwal C, Labuz J, Gabrys H (2013) Phosphoinositides play differential roles in regulating phototropin1- and phototropin2-mediated chloroplast movements in Arabidopsis. PLoS One 8:e55393

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Banas AK, Aggarwal C, Labuz J, Sztatelman O, Gabrys H (2012) Blue light signalling in chloroplast movements. J Exp Bot 63:1559–1574

    Article  CAS  PubMed  Google Scholar 

  • Berg R, Koniger M, Schjeide BM, Dikmak G, Kohler S, Harris GC (2006) A simple low-cost microcontroller-based photometric instrument for monitoring chloroplast movement. Photosynth Res 87:303–311

    Article  CAS  PubMed  Google Scholar 

  • Boersema PJ, Raijmakers R, Lemeer S, Mohammed S, Heck AJR (2009) Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat Protoc 4:484–494

    Article  CAS  PubMed  Google Scholar 

  • Boex-Fontvieille E, Daventure M, Jossier M, Zivy M, Hodges M, Tcherkez G (2013) Photosynthetic control of Arabidopsis leaf cytoplasmic translation initiation by protein phosphorylation. PLoS One 8:e70692

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Brett TJ, Traub LM, Fremont DH (2002) Accessory protein recruitment motifs in clathrin-mediated endocytosis. Structure 10:797–809

    Article  CAS  PubMed  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  CAS  PubMed  Google Scholar 

  • DeBlasio SL, Luesse DL, Hangarter RP (2005) A plant-specific protein essential for blue-light-induced chloroplast movements. Plant Physiol 139:101–114

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Demarsy E, Fankhauser C (2009) Higher plants use LOV to perceive blue light. Curr Opin Plant Biol 12:69–74

    Article  CAS  PubMed  Google Scholar 

  • Eisenberg E, Greene LE (2007) Multiple roles of auxilin and hsc70 in clathrin-mediated endocytosis. Traffic 8:640–646

    Article  CAS  PubMed  Google Scholar 

  • Ichikawa S, Yamada N, Suetsugu N, Wada M, Kadota A (2011) Red light, Phot1 and JAC1 modulate Phot2-dependent reorganization of chloroplast actin filaments and chloroplast avoidance movement. Plant Cell Physiol 52:1422–1432

    Article  CAS  PubMed  Google Scholar 

  • Inada S, Ohgishi M, Mayama T, Okada K, Sakai T (2004) RPT2 is a signal transducer involved in phototropic response and stomatal opening by association with phototropin 1 in Arabidopsis thaliana. Plant Cell 16:887–896

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Inoue S, Kinoshita T, Shimazaki K (2005) Possible involvement of phototropins in leaf movement of kidney bean in response to blue light. Plant Physiol 138:1994–2004

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Inoue S, Kinoshita T, Matsumoto M, Nakayama KI, Doi M, Shimazaki K (2008) Blue light-induced autophosphorylation of phototropin is a primary step for signalling. Proc Natl Acad Sci U S A 105:5626–5631

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Inoue S, Matsushita T, Tomokiyo Y, Matsumoto M, Nakayama KI, Kinoshita T, Shimazaki K (2011) Functional analyses of the activation loop of phototropin2 in Arabidopsis. Plant Physiol 156:117–128

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Jossier M, Kroniewicz L, Dalmas F, Le Thiec D, Ephritikhine G, Thomine S, Barbier-Brygoo H, Vavasseur A, Fillieur S, Leonhardt N (2010) The Arabidopsis vacuolar anion transporter, AtCLCc, is involved in the regulation of stomatal movements and contributes to salt tolerance. Plant J 64:563–576

    Google Scholar 

  • Kadota A, Yamada N, Suetsugu N, Hirose M, Saito C, Shoda K, Ichikawa S, Kagawa T, Nakano A, Wada M (2009) Short actin-based mechanism for light-directed chloroplast movement in Arabidopsis. Proc Natl Acad Sci U S A 106:13106–13111

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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  PubMed  Google Scholar 

  • Kaiserli E, Sullivan S, Jones MA, Feeney KA, Christie JM (2009) Domain swapping to assess the mechanistic basis of Arabidopsis phototropin 1 receptor kinase activation and endocytosis by blue light. Plant Cell 21:3226–3244

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kasahara M, Kagawa T, Oikawa K, Suetsugu N, Miyao M, Wada M (2002) Chloroplast avoidance movement reduces photodamage in plants. Nature 420:829–832

    Article  CAS  PubMed  Google Scholar 

  • 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  PubMed  Google Scholar 

  • Kodama Y, Suetsugu N, Kong SG, Wada M (2010) Two interacting coiled-coil proteins, WEB1 and PMI2, maintain the chloroplast photorelocation movement velocity in Arabidopsis. Proc Natl Acad Sci U S A 107:19591–19596

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kodama Y, Suetsugu N, Wada M (2011) Novel protein-protein interaction family proteins involved in chloroplast movement response. Plant Signal Behav 6:483–490

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kong LJ, Hanley-Bowdoin L (2002) A geminivirus replication protein interacts with a protein kinase and a motor protein that display different expression patterns during plant development and infection. Plant Cell 14:1817–1832

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kong SG, Wada M (2011) New insights into dynamic actin-based chloroplast photorelocation movement. Mol Plant 4:771–781

    Article  CAS  PubMed  Google Scholar 

  • Kong SG, Suzuki T, Tamura K, Mochizuki N, Hara-Nishimura I, Nagatani A (2006) Blue light-induced association of phototropin 2 with the Golgi apparatus. Plant J 45:994–1005

    Article  CAS  PubMed  Google Scholar 

  • Kong SG, Suetsugu N, Kikuchi S, Nakai M, Nagatani A, Wada M (2013) Both phototropin 1 and 2 localize on the chloroplast outer membrane with distinct localization activity. Plant Cell Physiol 54:80–92

    Article  CAS  PubMed  Google Scholar 

  • Lehmann P, Bohnsack MT, Schleiff E (2011) The functional domains of the chloroplast unusual positioning protein 1. Plant Sci 180:650–654

    Article  CAS  PubMed  Google Scholar 

  • Li P, Ainsworth EA, Leakey ADB, Ulanov A, Lozovaya V, Ort DR, Bohnert HJ (2008) Arabidopsis transcript and metabolite profiles: ecotype-specific responses to open-air elevated [CO2]. Plant Cell Environ 31:1673–1687

    Article  PubMed  Google Scholar 

  • Loreto F, Tsonev T, Centritto M (2009) The impact of blue light on leaf mesophyll conductance. J Exp Bot 60:2283–2290

    Article  CAS  PubMed  Google Scholar 

  • Luesse DR, DeBlasio SL, Hangarter RP (2006) Plastid movement impaired 2, a new gene involved in normal blue-light-induced chloroplast movements in Arabidopsis. Plant Physiol 141:1328–1337

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Miki H, Okada Y, Hirokawa N (2005) Analysis of the kinesin superfamily: insights into structure and function. Trends Cell Biol 15:467–476

    Article  CAS  PubMed  Google Scholar 

  • Motchoulski A, Liscum E (1999) Arabidopsis NPH3: a NPH1 photoreceptor-interacting protein essential for phototropism. Science 286:961–964

    Article  CAS  PubMed  Google Scholar 

  • Nakamura F, Amieva MR, Furthmayr H (1995) Phosphorylation of threonine 558 in the carboxyl-terminal actin-binding domain of moesin by thrombin activation of human platelets. J Biol Chem 270:31377–31385

    Article  CAS  PubMed  Google Scholar 

  • Nühse TS, Bottrill AR, Jones AME, Peck SC (2007) Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses. Plant J 51:931–940

    Article  PubMed Central  PubMed  Google Scholar 

  • Ohgishi M, Saji K, Okada K, Sakai T (2004) Functional analysis of each blue light receptor, cry1, cry2, phot1, and phot2, by using combinatorial multiple mutants in Arabidopsis. Proc Natl Acad Sci U S A 101:2223–2228

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Oikawa K, Kasahara M, Kiyosue T, Kagawa T, Suetsugu N, Takahashi F, Kanegae T, Niwa Y, Kadota A, Wada M (2003) Chloroplast unusual positioning1 is essential for proper chloroplast positioning. Plant Cell 15:2805–2815

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Pearson MA, Reczek D, Bretscher A, Karplus PA (2000) Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain. Cell 101:259–270

    Article  CAS  PubMed  Google Scholar 

  • Pedmale UV, Liscum E (2007) Regulation of phototropic signalling in Arabidopsis via phosphorylation state changes in the phototropin 1-interacting protein NPH3. J Biol Chem 282:19992–20001

    Article  CAS  PubMed  Google Scholar 

  • Pietromonaco SF, Simons PC, Altman A, Elias L (1998) Protein kinase C-theta phosphorylation of moesin in the actin-binding sequence. J Biol Chem 273:7594–7603

    Article  CAS  PubMed  Google Scholar 

  • Sakai T, Kagawa T, Kasahara M, Swartz TE, 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 U S A 98:6969–6974

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sakamoto K, Briggs WR (2002) Cellular and subcellular localization of phototropin 1. Plant Cell 14:1723–1735

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Salomon M, Eisenreich W, Durr H, Schleicher E, Knieb E, Massey V, Rudiger W, Muller F, Bacher A, Richter G (2001) An optomechanical transducer in the blue light receptor phototropin from Avena sativa. Proc Natl Acad Sci U S A 98:12357–12361

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Schmidt von Braun S, Schleiff E (2008) The chloroplast outer membrane protein CHUP1 interacts with actin and profilin. Planta 227:1151–1159

    Article  CAS  PubMed  Google Scholar 

  • Suetsugu N, Kagawa T, Wada M (2005) An auxilin-like J-domain protein, JAC1, regulates phototropin-mediated chloroplast movement in Arabidopsis. Plant Physiol 139:151–162

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Suetsugu N, Yamada N, Kagawa T, Yonekura H, Uyeda TQ, Kadota A, Wada M (2010) Two kinesin-like proteins mediate actin-based chloroplast movement in Arabidopsis thaliana. Proc Natl Acad Sci U S A 107:8860–8865

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sullivan S, Thomson CE, Lamont DJ, Jones MA, Christie JM (2008) In vivo phosphorylation site mapping and functional characterization of Arabidopsis phototropin 1. Mol Plant 1:178–194

    Article  CAS  PubMed  Google Scholar 

  • Sullivan S, Thomson CE, Kaiserli E, Christie JM (2009) Interaction specificity of Arabidopsis 14-3-3 proteins with phototropin receptor kinases. FEBS Lett 583:2187–2193

    Article  CAS  PubMed  Google Scholar 

  • 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  PubMed  Google Scholar 

  • Sztatelman O, Waloszek A, Banas AK, Gabrys H (2010) Photoprotective function of chloroplast avoidance movement: in vivo chlorophyll fluorescence study. J Plant Physiol 167:709–716

    Article  CAS  PubMed  Google Scholar 

  • Takamatsu H, Takagi S (2011) Actin-dependent chloroplast anchoring is regulated by Ca2+–calmodulin in spinach mesophyll cells. Plant Cell Physiol 52:1973–1982

    Article  CAS  PubMed  Google Scholar 

  • Tazoe Y, von Caemmerer S, Evans JR (2013) Measurement of mesophyll conductance in tobacco, Arabidopsis and wheat leaves with tunable diode laser absorption spectroscopy. Photosynth Res Food Fuel Future Springer 751–755

  • Tholen D, Boom C, Noguchi K, Ueda S, Katase T, Terashima I (2008) The chloroplast avoidance response decreases internal conductance to CO2 diffusion in Arabidopsis thaliana leaves. Plant Cell Environ 31:1688–1700

    Article  CAS  PubMed  Google Scholar 

  • Tsuboi H, Wada M (2011) Chloroplasts can move in any direction to avoid strong light. J Plant Res 124:201–210

    Article  PubMed  Google Scholar 

  • Tsuchida-Mayama T, Nakano M, Uehara Y, Sano M, Fujisawa N, Okada K, Sakai T (2008) Mapping of the phosphorylation sites on the phototropic signal transducer, NPH3. Plant Sci 174:626–633

    Article  CAS  Google Scholar 

  • Vanstraelen M, Torres Acosta JA, De Veylder L, Inze D, Geelen D (2004) A plant-specific subclass of C-terminal kinesins contains a conserved a-type cyclin-dependent kinase site implicated in folding and dimerization. Plant Physiol 135:1417–1429

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Vanstraelen M, Van Damme D, De Rycke R, Mylle E, Inze D, Geelen D (2006) Cell cycle-dependent targeting of a kinesin at the plasma membrane demarcates the division site in plant cells. Curr Biol 16:308–314

    Article  CAS  PubMed  Google Scholar 

  • Wada M (2013) Chloroplast movement. Plant Sci 210:177–182

    Article  CAS  PubMed  Google Scholar 

  • Wada M, Kong SG (2011) Analysis of chloroplast movement and relocation in Arabidopsis. Methods Mol Biol 774:87–102

    Article  CAS  PubMed  Google Scholar 

  • Walczak T, Gabrys H (1981) The carbon dioxide (CO2) effect on light-induced chloroplast translocations in higher plant leaves. Z Pflanzenphysiol 101:367–375

    Article  CAS  Google Scholar 

  • Wen F, Wang J, Xing D (2012) A protein phosphatase 2A catalytic subunit modulates blue light-induced chloroplast avoidance movements through regulating actin cytoskeleton in Arabidopsis. Plant Cell Physiol 53:1366–1379

    Article  CAS  PubMed  Google Scholar 

  • Whippo CW, Khurana P, Davis PA, DeBlasio SL, DeSloover D, Staiger CJ, Hangarter RP (2011) THRUMIN1 is a light-regulated actin-bundling protein involved in chloroplast motility. Curr Biol 21:59–64

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Eric Lambert and Jean-Paul Bares for their technical assistance, and Benjamin Roustan for his help in programming the microcontroller.

Funding

This work was supported by the Institut Fédératif de Recherche 87 for the phosphoproteomics analyses and the Agence Nationale de la Recherche for running costs (2 Jeunes Chercheurs grants (to G.T.), under contract nos. 08–330055 and 12–000101). E. B.-F. was supported by the Labex Saclay Plant Science through a post-doctoral grant.

Author information

Authors and Affiliations

  1. Institut de Biologie des Plantes, CNRS UMR 8618, Saclay Plant Sciences, Université Paris Sud, Orsay cedex, 91405, Paris, France

    Edouard Boex-Fontvieille, Mathieu Jossier, Michael Hodges & Guillaume Tcherkez

  2. Plateforme PAPPSO, UMR de Génétique Végétale, INRA/Université Paris Sud/CNRS/AgroParisTech, Ferme du Moulon, Gif-sur-Yvette, 91190, Paris, France

    Marlène Davanture & Michel Zivy

Authors
  1. Edouard Boex-Fontvieille
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mathieu Jossier
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marlène Davanture
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michel Zivy
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael Hodges
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Guillaume Tcherkez
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Edouard Boex-Fontvieille.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOCX 1495 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Boex-Fontvieille, E., Jossier, M., Davanture, M. et al. Differential Protein Phosphorylation Regulates Chloroplast Movement in Response to Strong Light and Darkness in Arabidopsis thaliana . Plant Mol Biol Rep 32, 987–1001 (2014). https://doi.org/10.1007/s11105-014-0707-3

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11105-014-0707-3

Keywords

Search

Navigation