Journal of Plant Research

, Volume 128, Issue 3, pp 407–421 | Cite as

Phytochrome-mediated regulation of cell division and growth during regeneration and sporeling development in the liverwort Marchantia polymorpha

  • Ryuichi NishihamaEmail author
  • Kimitsune Ishizaki
  • Masashi Hosaka
  • Yoriko Matsuda
  • Akane Kubota
  • Takayuki Kohchi
JPR Symposium Reprogramming of plant cells as adaptive strategies


Light regulates various aspects of development throughout the life cycle of sessile land plants. Photoreceptors, such as the red (R) and far-red (FR) light receptors phytochromes, play pivotal roles in modulating developmental programs. Reflecting high developmental plasticity, plants can regenerate tissues, organs, and whole bodies from varieties of cells. Among land plants, bryophytes exhibit extraordinary competency of regeneration under hormone-free conditions. As an environmental factor, light plays critical roles in regeneration of bryophytes. However, how light regulates regeneration remains unknown. Here we show that using the liverwort Marchantia polymorpha, which contains a single phytochrome gene, the phytochrome regulates re-entry into the cell cycle and cell shape in newly regenerating tissues. Our morphological and cytological observations revealed that S-phase entry of G1-arrested epidermal cells around the midrib on the ventral surface of thallus explants was greatly retarded in the dark or under phytochrome-inactive R/FR cycle irradiation conditions, where, nevertheless, small, laterally narrow regenerants were eventually formed. Thus, consistent with earlier descriptions published over a century ago, light is not essential for, but exerts profound effects on regeneration in M. polymorpha. Ventral cells in regenerants grown under R/FR cycle conditions were longer and narrower than those under R cycle. Expression of a constitutively active mutant of M. polymorpha phytochrome allowed regeneration of well grown, widely expanded thalli even in the dark when sugar was supplied, further demonstrating that the phytochrome signal promotes cell proliferation, which is rate-limited by sucrose availability. Similar effects of R and FR irradiation on cell division and elongation were observed in sporelings as well. Thus, besides activation of photosynthesis, major roles of R in regeneration of M. polymorpha are to facilitate proliferation of rounder cells through the phytochrome by mechanisms that are likely to operate in the sporeling.


Cell division cycle Cell shape Dedifferentiation Phytochrome Regeneration Sugar signaling 



We thank John Bowman for information on classical literature and helpful discussion, Tom Dierschke for translating German literature, Sachihiro Matsunaga for the EdU technique, and Keisuke Inoue for experimental supports and discussion. This work was supported by KAKENHI Grant-in-Aids for Scientific Research on Innovative Area (Nos. 23120516 and 25113009 to T.K.), for Young Scientists (B) (No. 22770035 to K.I.), and for Scientific Research (C) (No. 24570048 to R. N.) from the Japan Society for the Promotion of Science.

Supplementary material

10265_2015_724_MOESM1_ESM.pdf (1.7 mb)
Fig. S1 Apical-basal polarity in regeneration. a Schematic illustration of excision. A thallus of M. polymorpha (thin green line) was excised with a scalpel along the red broken lines. Thick green line midrib. b, c Micrographs of explants. Both apical and basal explants (b) were incubated for 5 days on sugar-free medium (c). Arrows regenerants. Bars 2 mm. Fig. S2 Schematic illustrations of excision. Pink-shaded fragments were used for the experiments shown in Figs. 2, S3, S5 (a) and Figs. 4–6, S7 (b). The excision pattern in a was used for SEM analyses to make sure that only one midrib is included in an explant. Fig. S3 SEM observation of the ventral side of a thallus and explant. a, b Ventral view of a 12-day-old thallus. Magnified view of the boxed region in a is shown in b. The dotted line marks a typical excision position. pr pegged rhizoid, sc scale, sr smooth rhizoid. c Ventral view of an explant 72 h after excision. Bars 500 µm (a, c), 100 µm (b). Fig. S4 Light irradiation patterns for R and R/FR cycle conditions used in this study. Fig. S5 SEM observation of initial stages in regeneration under various light conditions. Explants were grown for 96 h in the dark (a, d) or under R (b, e) or R/FR cycle (c, f) in the absence (ac) or presence (df) of 1 % sucrose. Ventral-side views are presented. Apical side of each explant is shown upside. Bars 100 µm. Fig. S6 An amino-acid sequence alignment of GAF domains. The alignment of GAF domains in PHY from M. polymorpha and PHYB from Arabidopsis was constructed using the MUSCLE program [Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797] implemented in the Geneious software (version 6.1.8; Biomatters; with default parameters. The conserved tyrosine residue that was substituted to histidine is marked by triangle. Black shade identical residues. Fig. S7 Comparison of morphology of regenerants grown under R or R/FR cycle. Explants of 10-day-old thalli were incubated on sugar-free media under R (a, b) or R/FR cycle (c, d) for 2 weeks. a, c Dorsal, side, and ventral views of regenerants. b, d Transverse sections of agar-embedded regenerants dissected approximately along the broken lines in a and c. Red arrow air chamber. Bars 200 µm (a, c), 100 µm (b, d).(PDF 1729 kb)
10265_2015_724_MOESM2_ESM.mpg (5.9 mb)
Supplemental Movie 1 Complete series of time-lapse observation of regeneration for 5 days from a thallus explant of M. polymorpha. A basal fragment obtained from a 14-day-old thallus of Tak-1 was placed on solid medium containing 1 % sucrose and incubated under continuous white light. Photographs were taken with 1-h intervals.(MPG 6 044 kb)


  1. Althoff F, Kopischke S, Zobell O, Ide K, Ishizaki K, Kohchi T, Zachgo S (2014) Comparison of the MpEF1α and CaMV35 promoters for application in Marchantia polymorpha overexpression studies. Transgenic Res 23:235–244CrossRefPubMedGoogle Scholar
  2. Bauer L, Mohr H (1959) Der Nachweis des reversiblen Hellrot-Dunkelrot-Reaktions systems bei Laubmoosen. Planta 54:68–73CrossRefGoogle Scholar
  3. Brücker G, Mittmann F, Hartmann E, Lamparter T (2005) Targeted site-directed mutagenesis of a heme oxygenase locus by gene replacement in the moss Ceratodon purpureus. Planta 220:864–874CrossRefPubMedGoogle Scholar
  4. Casal JJ (2013) Photoreceptor signaling networks in plant responses to shade. Annu Rev Plant Biol 64:403–427CrossRefPubMedGoogle Scholar
  5. Cavers F (1903) On asexual reproduction and regeneration in Hepaticae. New Phytol 2:121–133CrossRefGoogle Scholar
  6. Christie JM (2007) Phototropin blue-light receptors. Annu Rev Plant Biol 58:21–45CrossRefPubMedGoogle Scholar
  7. Cove DJ, Schild A, Ashton NW, Hartmann E (1978) Genetic and physiological studies of the effect of light on the development of the moss, Physcomitrella patens. Photochem Photobiol 27:249–254CrossRefGoogle Scholar
  8. Dickson H (1932) Polarity and the production of adventitious growing points in Marchantia polymorpha. Ann Bot 46:683–684Google Scholar
  9. Fraikin GY, Strakhovskaya MG, Rubin AB (2013) Biological photoreceptors of light-dependent regulatory processes. Biochemistry (Mosc) 78:1238–1253CrossRefGoogle Scholar
  10. Franklin KA, Quail PH (2010) Phytochrome functions in Arabidopsis development. J Exp Bot 61:11–24CrossRefPubMedCentralPubMedGoogle Scholar
  11. Furuya M, Kanno M, Okamoto H, Fukuda S, Wada M (1997) Control of mitosis by phytochrome and a blue-light receptor in fern spores. Plant Physiol 113:677–683PubMedCentralPubMedGoogle Scholar
  12. Galbraith DW (2009) Simultaneous flow cytometric quantification of plant nuclear DNA contents over the full range of described angiosperm 2C values. Cytometry A 75:692–698CrossRefPubMedGoogle Scholar
  13. Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, Firoozabady E (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220:1049–1051CrossRefPubMedGoogle Scholar
  14. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158CrossRefPubMedGoogle Scholar
  15. Giles KL (1971) Dedifferentiation and regeneration in bryophytes: a selective review. NZ J Bot 9:689–694CrossRefGoogle Scholar
  16. Giles KL, von Maltzahn KE (1967) Interaction of red, far-red, and blue light in cellular regeneration of leaves of Mnium affine. Bryologist 70:312–315CrossRefGoogle Scholar
  17. Goebel K (1898) Archegoniatenstudien. VIII. Rückschlagsbildungen und Sprossung bei Metzgeria. Flora 85:69–74Google Scholar
  18. Goebel K (1907) Archegoniatenstudien. XI. Weitere Untersuchungen über Keimung und Regeneration bei Riella und Sphaerocarpus. Flora 97:192–215Google Scholar
  19. Hansel V (1876) Über die Keimung der Preissia commutata N. v. E. Sitzungsber Kais Akad Wissensch (Wien) 73:89–97Google Scholar
  20. Hartmann E, Klingenberg B, Bauer L (1983) Phytochrome-mediated phototropism in protonemata of the moss Ceratodon purpureus Brid. Photochem Photobiol 38:599–603CrossRefGoogle Scholar
  21. Heald FdF (1898a) Conditions for the germination of the spores of bryophytes and pteridophytes. Bot Gaz 26:25–45CrossRefGoogle Scholar
  22. Heald FdF (1898b) A study of regeneration as exhibited by mosses. Bot Gaz 26:169–210CrossRefGoogle Scholar
  23. Hu W, Su YS, Lagarias JC (2009) A light-independent allele of phytochrome B faithfully recapitulates photomorphogenic transcriptional networks. Mol Plant 2:166–182CrossRefPubMedCentralPubMedGoogle Scholar
  24. Ishikawa M, Hasebe M (2015) Cell cycle reentry from the late S phase: implications from stem cell formation in the moss Physcomitrella patens. J Plant Res (this issue). doi: 10.1007/s10265-015-0713-z
  25. Ishikawa M, Murata T, Sato Y, Nishiyama T, Hiwatashi Y, Imai A, Kimura M, Sugimoto N, Akita A, Oguri Y, Friedman WE, Hasebe M, Kubo M (2011) Physcomitrella cyclin-dependent kinase A links cell cycle reactivation to other cellular changes during reprogramming of leaf cells. Plant Cell 23:2924–2938CrossRefPubMedCentralPubMedGoogle Scholar
  26. Ishizaki K, Chiyoda S, Yamato KT, Kohchi T (2008) Agrobacterium-mediated transformation of the haploid liverwort Marchantia polymorpha L., an emerging model for plant biology. Plant Cell Physiol 49:1084–1091CrossRefPubMedGoogle Scholar
  27. Ishizaki K, Johzuka-Hisatomi Y, Ishida S, Iida S, Kohchi T (2013) Homologous recombination-mediated gene targeting in the liverwort Marchantia polymorpha L. Sci Rep 3:1532PubMedCentralPubMedGoogle Scholar
  28. Ito S, Song YH, Imaizumi T (2012) LOV domain-containing F-box proteins: light-dependent protein degradation modules in Arabidopsis. Mol Plant 5:573–582CrossRefPubMedGoogle Scholar
  29. Jenkins GI, Cove DJ (1983) Light requirements for regeneration of protoplasts of the moss Physcomitrella patens. Planta 157:39–45CrossRefPubMedGoogle Scholar
  30. Kadota A, Wada M, Furuya M (1982) Phytochrome-mediated phototropism and different dichroic orientation of Pr and Pfr in protonemata of the fern Adiantum capillus-veneris L. Photochem Photobiol 35:533–536CrossRefGoogle Scholar
  31. Kami C, Lorrain S, Hornitschek P, Fankhauser C (2010) Light-regulated plant growth and development. In: Marja CPT (ed) Current Topics in Developmental Biology, vol 91. Academic Press, Oxford, UK, pp 29–66Google Scholar
  32. Kaul KN, Mitra GC, Tripathi BK (1962) Responses of Marchantia in aseptic culture to well-known auxins and antiauxins. Ann Bot 26:447–466Google Scholar
  33. 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 166:411–427CrossRefPubMedGoogle Scholar
  34. Kreh W (1909) Über die Regeneration der Lebermoose. Karras, HalleGoogle Scholar
  35. 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
  36. Kubota A, Kita S, Ishizaki K, Nishihama R, Yamato KT, Kohchi T (2014) Co-option of a photoperiodic growth-phase transition system during land plant evolution. Nat Commun 5:3668CrossRefPubMedGoogle Scholar
  37. Liu H, Liu B, Zhao C, Pepper M, Lin C (2011) The action mechanisms of plant cryptochromes. Trends Plant Sci 16:684–691CrossRefPubMedCentralPubMedGoogle Scholar
  38. López-Juez E, Dillon E, Magyar Z, Khan S, Hazeldine S, de Jager SM, Murray JA, Beemster GT, Bögre L, Shanahan H (2008) Distinct light-initiated gene expression and cell cycle programs in the shoot apex and cotyledons of Arabidopsis. Plant Cell 20:947–968CrossRefPubMedCentralPubMedGoogle Scholar
  39. Mittmann F, Brücker G, Zeidler M, Repp A, Abts T, Hartmann E, Hughes J (2004) Targeted knockout in Physcomitrella reveals direct actions of phytochrome in the cytoplasm. Proc Natl Acad Sci USA 101:13939–13944CrossRefPubMedCentralPubMedGoogle Scholar
  40. Nakazato T, Kadota A, Wada M (1999) Photoinduction of spore germination in Marchantia polymorpha L. is mediated by photosynthesis. Plant Cell Physiol 40:1014–1020CrossRefGoogle Scholar
  41. Necker NJd (1774) Physiologia Museorum. Manhemii, SchwanGoogle Scholar
  42. Neff MM, Fankhauser C, Chory J (2000) Light: an indicator of time and place. Genes Dev 14:257–271PubMedGoogle Scholar
  43. Nishihama R, Kohchi T (2013) Evolutionary insights into photoregulation of the cell cycle in the green lineage. Curr Opin Plant Biol 16:630–637CrossRefPubMedGoogle Scholar
  44. Okada S, Fujisawa M, Sone T, Nakayama S, Nishiyama R, Takenaka M, Yamaoka S, Sakaida M, Kono K, Takahama M, Yamato KT, Fukuzawa H, Brennicke A, Ohyama K (2000) Construction of male and female PAC genomic libraries suitable for identification of Y-chromosome-specific clones from the liverwort, Marchantia polymorpha. Plant J 24:421–428CrossRefPubMedGoogle Scholar
  45. Prigge MJ, Bezanilla M (2010) Evolutionary crossroads in developmental biology: Physcomitrella patens. Development 137:3535–3543CrossRefPubMedGoogle Scholar
  46. Rickett HW (1920) Regeneration in Sphaerocarpos Donnellii. Bull Torrey Bot Club 47:347–357CrossRefGoogle Scholar
  47. Riou-Khamlichi C, Menges M, Healy JMS, Murray JAH (2000) Sugar control of the plant cell cycle: differential regulation of Arabidopsis D-type cyclin gene expression. Mol Cell Biol 20:4513–4521CrossRefPubMedCentralPubMedGoogle Scholar
  48. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675CrossRefPubMedGoogle Scholar
  49. Schostakowitsch W (1894) Ueber die Reproductions-und Regenerations erscheinungen bei den Lebermoosen. Flora 79:350–384Google Scholar
  50. Soni R, Carmichael JP, Shah ZH, Murray JA (1995) A family of cyclin D homologs from plants differentially controlled by growth regulators and containing the conserved retinoblastoma protein interaction motif. Plant Cell 7:85–103CrossRefPubMedCentralPubMedGoogle Scholar
  51. Su YS, Lagarias JC (2007) Light-independent phytochrome signaling mediated by dominant GAF domain tyrosine mutants of Arabidopsis phytochromes in transgenic plants. Plant Cell 19:2124–2139CrossRefPubMedCentralPubMedGoogle Scholar
  52. Sugano SS, Shirakawa M, Takagi J, Matsuda Y, Shimada T, Hara-Nishimura I, Kohchi T (2014) CRISPR/Cas9-mediated targeted mutagenesis in the liverwort Marchantia polymorpha L. Plant Cell Physiol 55:475–481CrossRefPubMedGoogle Scholar
  53. Sugimoto K, Jiao Y, Meyerowitz EM (2010) Arabidopsis regeneration from multiple tissues occurs via a root development pathway. Dev Cell 18:463–471CrossRefPubMedGoogle Scholar
  54. Valanne N (1966) The germination phases of moss spores and their control by light. Ann Bot Fenn 3:1Google Scholar
  55. Vöchting H (1885) Über die regeneration der Marchantieen. Jahrbücher für wissenschaftliche Botanik 16:367–414Google Scholar
  56. Wada M (1985) Photoresponses in cell cycle regulation. Proc R Soc Edinb Biol 86:231–235Google Scholar
  57. Wada M, Kadota A (1989) Photomorphogenesis in lower green plants. Annu Rev Plant Physiol Plant Mol Biol 40:169–191CrossRefGoogle Scholar
  58. Wada M, Hayami J, Kadota A (1984) Returning dark-induced cell cycle to the beginning of G1 phase by red light irradiation in fern Adiantum protonemata. Plant Cell Physiol 25:1053–1058Google Scholar
  59. Xiong Y, McCormack M, Li L, Hall Q, Xiang C, Sheen J (2013) Glucose-TOR signalling reprograms the transcriptome and activates meristems. Nature 496:181–186CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© The Botanical Society of Japan and Springer Japan 2015

Authors and Affiliations

  • Ryuichi Nishihama
    • 1
    Email author
  • Kimitsune Ishizaki
    • 1
    • 2
  • Masashi Hosaka
    • 1
  • Yoriko Matsuda
    • 1
  • Akane Kubota
    • 1
    • 3
  • Takayuki Kohchi
    • 1
  1. 1.Graduate School of BiostudiesKyoto UniversityKyotoJapan
  2. 2.Graduate School of ScienceKobe UniversityKobeJapan
  3. 3.Department of BiologyUniversity of WashingtonSeattleUSA

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