Journal of Plant Research

, Volume 130, Issue 6, pp 1061–1070 | Cite as

Chloroplast aggregation during the cold-positioning response in the liverwort Marchantia polymorpha

  • Hiroyuki Tanaka
  • Mayuko Sato
  • Yuka Ogasawara
  • Noriko Hamashima
  • Othmar Buchner
  • Andreas Holzinger
  • Kiminori Toyooka
  • Yutaka KodamaEmail author
Regular Paper


Under low-light conditions, chloroplasts localize along periclinal cell walls at temperatures near 20 °C, but they localize along anticlinal cell walls near 5 °C. This phenomenon is known as the cold-positioning response. We previously showed that chloroplasts move as aggregates rather than individually during the cold-positioning response in the fern Adiantum capillus-veneris. This observation suggested that chloroplasts physically interact with each other during the cold-positioning response. However, the physiological processes underlying chloroplast aggregation are unclear. In this report, we characterized chloroplast aggregation during the cold-positioning response in the liverwort Marchantia polymorpha. Confocal laser microscopy observations of transgenic liverwort plants expressing a fluorescent fusion protein that localizes to the chloroplast outer envelope membrane (OEP7-Citrine) showed that neighboring chloroplast membranes did not fuse during the cold-positioning response. Transmission electron microscopy analysis revealed that a distance of at least 10 nm was maintained between neighboring chloroplasts during aggregation. These results indicate that aggregated chloroplasts do not fuse, but maintain a distance of at least 10 nm from each other during the cold-positioning response.


Bryophytes Chloroplast aggregation Chloroplast movement Low temperature Outer envelope membrane Temperature-controlled microscopy 



We thank Mr. Koichiro Takaoka (THERMTRON Co. Ltd., Japan) and Mr. Tadao Onuma (Onuma Factory Co. Ltd., Japan; Study Group of Ohtawara Health and Welfare) for their help in developing the temperature-controlled microscope system. We also thank Dr. Takayuki Kohchi (Kyoto University) for providing the Tak-1 and BC3-38 strains. H.T. was supported by a Postdoctoral Fellowship of the Creative Department for Innovation of Utsunomiya University. N.H. was supported by the Hayashi Rheology Memorial Foundation. This work was supported by a Fellowship for Overseas Collaboration Research of the Japanese Society of Plant Physiologists (JSPP) (for collaboration between A.H. and Y.K.), the Japan Society for the Promotion of Science (JSPS) KAKENHI (nos. 23870002 and 26840088 to Y.K.), the JST-ERATO Numata Organelle Reaction Cluster (JPMJER1602 to Y.K.), and Research and Development Grants of the Creative Department for Innovation of Utsunomiya University (Y.K.).

Supplementary material

10265_2017_958_MOESM1_ESM.pdf (8.6 mb)
Supplementary material 1 (PDF 8782 KB)
10265_2017_958_MOESM2_ESM.avi (3.8 mb)
Movie S1: Time-lapse observation of chloroplast movement during the avoidance response induced by high-intensity white light. Images were acquired at 1-min intervals for 5 h. Bar = 5 μm. (AVI 3928 KB)
10265_2017_958_MOESM3_ESM.avi (1.5 mb)
Movie S2: Time-lapse observation of chloroplast movement during the cold-positioning response under low-intensity white light conditions. Images were acquired at 5-min intervals for 12 h. Bar = 5 μm. (AVI 1534 KB)


  1. Bannai H, Tamada Y, Maruyama O, Nakai K, Miyano S (2002) Extensive feature detection of N-terminal protein sorting signals. Bioinformatics 18:298–305CrossRefPubMedGoogle Scholar
  2. Bowman JL, Araki T, Kohchi T (2016) Marchantia: past, present and future. Plant Cell Physiol 57:205–209CrossRefPubMedGoogle Scholar
  3. Buchner O, Lütz C, Holzinger A (2007) Design and construction of a new temperature-controlled chamber for light and confocal microscopy under monitored conditions: biological application for plant samples. J Microsc 225:183–191CrossRefPubMedGoogle Scholar
  4. Gabryś H, Walczak T, Malec P (1997) Interaction between phytochrome and blue light photoreceptor system in Mougeotia: temperature dependence. J Photochem Photobiol B 38:35–39CrossRefGoogle Scholar
  5. Haupt W, Mortel G, Winkelnkemper I (1969) Demonstration of different dichroic orientation of phytochrome Pr and Pfr. Planta 88:183–186CrossRefPubMedGoogle Scholar
  6. Inoue K (2007) The chloroplast outer envelope membrane: the edge of light and excitement. J Integr Plant Biol 49:1100–1111CrossRefGoogle Scholar
  7. Ishizaki K, Nishihama R, Ueda M, Inoue K, Ishida S, Nishimura Y, Shikanai T, Kohchi T (2015) Development of gateway binary vector series with four different selection markers for the liverwort Marchantia polymorpha. PLoS One 10:e0138876CrossRefPubMedPubMedCentralGoogle Scholar
  8. Kadota A, Sato Y, Wada M (2000) Intracellular chloroplast photorelocation in the moss Physcomitrella patens is mediated by phytochrome as well as by a blue-light receptor. Planta 210:932–937CrossRefPubMedGoogle Scholar
  9. Kadowaki Y, Sato Y, Ghosh TK, Takezawa D (2015) Inhibition by abscisic acid of cold-induced relocation of chloroplasts in the liverwort Marchantia polymorpha. Cryobiol Cryotechnol 61:145–150Google Scholar
  10. Kagawa T, Wada M (1994) Brief irradiation with red or blue light induces orientational movement of chloroplasts in dark-adapted prothallial cells of the fern Adiantum. J Plant Res 107:389–398CrossRefGoogle Scholar
  11. Kagawa T, Wada M (1996) Phytochrome- and blue-light-absorbing pigment-mediated directional movement of chloroplasts in dark-adapted prothallial cells of fern Adiantum as analyzed by microbeam irradiation. Planta 198:488–493CrossRefGoogle Scholar
  12. Kagawa T, Wada M (1999) Chloroplast avoidance response induced by blue light of high fluence rate in prothallial cells of the fern Adiantum as analyzed by microbeam irradiation. Plant Physiol 119:917–923CrossRefPubMedPubMedCentralGoogle Scholar
  13. Kagawa T, Lampater T, Hartmann E, Wada M (1997) Phytochrome mediated branch formation in moss Ceratodon. J Plant Res 110:363–370CrossRefGoogle Scholar
  14. 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 291:2138–2141CrossRefPubMedGoogle Scholar
  15. Kasahara M, Kagawa T, Oikawa K, Suetsugu N, Miyao M, Wada M (2002) Chloroplast avoidance movement reduces photodamage in plants. Nature 420:829–832CrossRefPubMedGoogle Scholar
  16. Kimura S, Kodama Y (2016) Actin-dependence of the chloroplast cold positioning response in the liverwort Marchantia polymorpha L. PeerJ 4:e2513CrossRefPubMedPubMedCentralGoogle Scholar
  17. Kodama Y (2016) Time gating of chloroplast autofluorescence allows clearer fluorescence imaging in planta. PLoS One 11:e0152484CrossRefPubMedPubMedCentralGoogle Scholar
  18. Kodama Y, Tsuboi H, Kagawa T, Wada M (2008) Low temperature-induced chloroplast relocation mediated by a blue light receptor, phototropin 2, in fern gametophytes. J Plant Res 121:441–448CrossRefPubMedGoogle Scholar
  19. Komatsu A, Terai M, Ishizaki K, Suetsugu N, Tsuboi H, Nishihama R, Yamato KT, Wada M, Kohchi T (2014) Phototropin encoded by a single-copy gene mediates chloroplast photorelocation movements in the liverwort Marchantia polymorpha. Plant Physiol 166:411–427CrossRefPubMedPubMedCentralGoogle Scholar
  20. Kondo A, Kaikawa J, Funaguma T, Ueno O (2004) Clumping and dispersal of chloroplasts in succulent plants. Planta 219:500–506CrossRefPubMedGoogle Scholar
  21. Kraml M, Biittner G, Haupt W, Herrmann H (1988) Chloroplast orientation in Mesotaenium: the phytochrome effect is strongly potentiated by interaction with blue light. Protoplasma Suppl 1:172–179CrossRefGoogle Scholar
  22. Kubota A, Ishizaki K, Hosaka M, Kohchi T (2013) Efficient Agrobacterium-mediated transformation of the liverwort Marchantia polymorpha using regenerating thalli. Biosci Biotechnol Biochem 77:167–172CrossRefPubMedGoogle Scholar
  23. Łabuz J, Hermanowicz P, Gabryś H (2015) The impact of temperature on blue light induced chloroplast movements in Arabidopsis thaliana. Plant Sci 239:238–249CrossRefPubMedGoogle Scholar
  24. Lee YJ, Kim DH, Kim YW, Hwang I (2001) Identification of a signal that distinguishes between the chloroplast outer envelope membrane and the endomembrane system in vivo. Plant Cell 13:2175–2190CrossRefPubMedPubMedCentralGoogle Scholar
  25. Ogasawara Y, Ishizaki K, Kohchi T, Kodama Y (2013) Cold-induced organelle relocation in the liverwort Marchantia polymorpha L. Plant Cell Environ 36:1520–1528CrossRefPubMedGoogle Scholar
  26. 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 15:2805–2815CrossRefPubMedPubMedCentralGoogle Scholar
  27. Oikawa K, Yamasato A, Kong SG, Kasahara M, Nakai M, Takahashi F, Ogura Y, Kagawa T, Wada M (2008) Chloroplast outer envelope protein CHUP1 is essential for chloroplast anchorage to the plasma membrane and chloroplast movement. Plant Physiol 148:829–842CrossRefPubMedPubMedCentralGoogle Scholar
  28. Ono K, Ohyama K, Gamborg OL (1979) Regeneration of the liverwort Marchantia polymorpha L. from protoplasts isolated from cell suspension culture. Plant Sci Lett 14:225–229CrossRefGoogle Scholar
  29. Qiu YL, Li L, Wang B, Chen Z, Knoop V, Groth-Malonek M, Dombrovska O, Lee J, Kent L, Rest J, Estabrook GF, Hendry TA, Taylor DW, Testa CM, Ambros M, Crandall-Stotler B, Duff RJ, Stech M, Frey W, Quandt D, Davis CC (2006) The deepest divergences in land plants inferred from phylogenomic evidence. Proc Natl Acad Sci USA 103:15511–15516CrossRefPubMedPubMedCentralGoogle Scholar
  30. Rasband WS (1997–2016) ImageJ. U.S. National Institutes of Health, Bethesda.
  31. Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208–212CrossRefPubMedPubMedCentralGoogle Scholar
  32. Schattat MH, Griffiths S, Mathur N, Barton K, Wozny MR, Dunn N, Greenwood JS, Mathur J (2012) Differential coloring reveals that plastids do not form networks for exchanging macromolecules. Plant Cell 4:1465–1477CrossRefGoogle Scholar
  33. Senn G (1908) Die Gestalts—und Lageveränderung der Pflanzen—Chromatophoren. Wilhelm-Engelmann, LeipzigGoogle Scholar
  34. Shen Z, Liu YC, Bibeau JP, Lemoi KP, Tüzel E, Vidali L (2015) The kinesin-like proteins, KAC1/2, regulate actin dynamics underlying chloroplast light-avoidance in Physcomitrella patens. J Integr Plant Biol 57:106–119CrossRefPubMedGoogle Scholar
  35. Suetsugu N, Kagawa T, Wada M (2005) An auxilin-like J-domain protein, JAC1, regulates phototropin-mediated chloroplast movement in Arabidopsis. Plant Physiol 139:151–162CrossRefPubMedPubMedCentralGoogle Scholar
  36. 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 USA 107:8860–8865CrossRefPubMedPubMedCentralGoogle Scholar
  37. Suetsugu N, Sato Y, Tsuboi H, Kasahara M, Imaizumi T, Kagawa T, Hiwatashi Y, Hasebe M, Wada M (2012) The KAC family of kinesin-like proteins is essential for the association of chloroplasts with the plasma membrane in land plants. Plant Cell Physiol 53:1854–1865CrossRefPubMedGoogle Scholar
  38. Thevenaz P, Ruttimann UE, Unser M (1998) A pyramid approach to subpixel registration based on intensity. IEEE Trans Image Proc 7:27–41CrossRefGoogle Scholar
  39. Tsuboyama-Tanaka S, Kodama Y (2015) AgarTrap-mediated genetic transformation using intact gemmae/gemmalings of the liverwort Marchantia polymorpha L. J Plant Res 128:337–344CrossRefPubMedGoogle Scholar
  40. von Braun SS, Schleiff E (2008) The chloroplast outer membrane protein CHUP1 interacts with actin and profilin. Planta 227:1151–1159CrossRefGoogle Scholar
  41. Wada M, Kagawa T, Sato Y (2003) Chloroplast movement. Annu Rev Plant Biol 54:455–468CrossRefPubMedGoogle Scholar
  42. Yamada M, Kawasaki M, Sugiyama T, Miyake H, Taniguchi M (2009) Differential positioning of C4 mesophyll and bundle sheath chloroplasts: aggregative movement of C4 mesophyll chloroplasts in response to environmental stresses. Plant Cell Physiol 50:1736–1749CrossRefPubMedGoogle Scholar
  43. Yatsuhashi H, Kobayashi H (1993) Dual involvement of phytochrome in light-oriented chloroplast movement in Dryopteris sparsa protonemata. Photochem Photobiol B 19:25–31CrossRefGoogle Scholar
  44. Zurzycki J, Lelatko Z (1969) Action dichroism in the chloroplast rearrangements in various plant species. Acta Soc Bot Poloniae 38:493–506CrossRefGoogle Scholar

Copyright information

© The Botanical Society of Japan and Springer Japan KK 2017

Authors and Affiliations

  • Hiroyuki Tanaka
    • 1
    • 2
  • Mayuko Sato
    • 3
  • Yuka Ogasawara
    • 1
  • Noriko Hamashima
    • 1
  • Othmar Buchner
    • 4
  • Andreas Holzinger
    • 4
  • Kiminori Toyooka
    • 3
  • Yutaka Kodama
    • 1
    Email author
  1. 1.Center for Bioscience Research and EducationUtsunomiya UniversityTochigiJapan
  2. 2.Collaboration Center for Research and DevelopmentUtsunomiya UniversityTochigiJapan
  3. 3.Center for Sustainable Resource Science, RIKENKanagawaJapan
  4. 4.Institute of BotanyUniversity of InnsbruckInnsbruckAustria

Personalised recommendations