Ectopic Vascular Induction in Arabidopsis Cotyledons for Sequential Analysis of Phloem Differentiation

  • Alif Meem Nurani
  • Yuki KondoEmail author
  • Hiroo FukudaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1830)


Vascular system is vital for the transport of water and nutrients as well as for providing mechanical support in many land plants. Plant transcription factors play a central role in regulating vascular development downstream of hormones and peptide signaling pathways. Particularly, cell culture systems have contributed to isolating such key transcription factors for xylem differentiation. However, there had been no efficient systems that can mimic phloem differentiation in the model plant Arabidopsis, preventing the identification of phloem-related transcription factors. We have recently established Vascular cell Induction culture System Using Arabidopsis Leaves (VISUAL), which concomitantly generates both xylem and phloem cells in the cotyledon of Arabidopsis. This system can be used to take a closer look at the bi-directional differentiation mechanism of (pro)cambial cells into xylem and phloem cells. Here, we report the methods of microscopic, genetic, and molecular analysis using VISUAL, which can help in decrypting the transcriptional networks that regulate vascular cell differentiation.

Key words

Vascular induction Arabidopsis Differentiation Procambium Cambium Xylem Phloem Cell-sorting Tissue culture 



This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (17H06476 to Y.K. and 15H05958 to H.F.), from the Japan Society for the Promotion of Science (17H05008 to Y.K. and 16H06377 to H.F.) and from Naito Foundation to H.F.


  1. 1.
    Baima S, Possenti M, Matteucci A, Wisman E, Altamura MM, Ruberti I et al (2001) The arabidopsis ATHB-8 HD-zip protein acts as a differentiation-promoting transcription factor of the vascular meristems. Plant Physiol 126:643–655CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Bonke M, Thitamadee S, Mähönen AP, Hauser MT, Helariutta Y (2003) APL regulates vascular tissue identity in Arabidopsis. Nature 426:181–186CrossRefPubMedGoogle Scholar
  3. 3.
    Emery JF, Floyd SK, Alvarez J, Eshed Y, Hawker NP, Izhaki A et al (2003) Radial patterning of Arabidopsis shoots by class III HD-ZIP and KANADI genes. Curr Biol 13:1768–1774CrossRefPubMedGoogle Scholar
  4. 4.
    Kubo M, Udagawa M, Nishikubo N, Horiguchi G, Yamaguchi M, Ito J et al (2005) Transcription switches for protoxylem and metaxylem vessel formation. Genes Dev 19:1855–1860CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Mitsuda N, Iwase A, Yamamoto H, Yoshida M, Seki M, Shinozaki K et al (2007) NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis. Plant Cell 19:270–280CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Furuta KM, Yadav SR, Lehesranta S, Belevich I, Miyashima S, Heo JO et al (2014) Plant development. Arabidopsis NAC45/86 direct sieve element morphogenesis culminating in enucleation. Science 345:933–937CrossRefPubMedGoogle Scholar
  7. 7.
    Wallner ES, López-Salmerón V, Belevich I, Poschet G, Jung I, Grünwald K et al (2017) Strigolactone- and karrikin-independent SMXL proteins are central regulators of phloem formation. Curr Biol 27:1241–1247CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Fukuda H, Komamine A (1980) Establishment of an experimental system for the study of tracheary element differentiation from single cells isolated from the mesophyll of zinnia elegans. Plant Physiol 65:57–60CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Motose H, Sugiyama M, Fukuda H (2004) A proteoglycan mediates inductive interaction during plant vascular development. Nature 429:873–878CrossRefPubMedGoogle Scholar
  10. 10.
    Ito Y, Nakanomyo I, Motose H, Iwamoto K, Sawa S, Dohmae N et al (2006) Dodeca-CLE peptides as suppressors of plant stem cell differentiation. Science 313:842–845CrossRefGoogle Scholar
  11. 11.
    Oda Y, Iida Y, Kondo Y, Fukuda H (2010) Wood cell-wall structure requires local 2D-microtubule disassembly by a novel plasma membrane-anchored protein. Curr Biol 20:1197–1202CrossRefPubMedGoogle Scholar
  12. 12.
    Pesquet E, Korolev AV, Calder G, Lloyd CW (2010) The microtubule-associated protein AtMAP70-5 regulates secondary wall patterning in Arabidopsis wood cells. Curr Biol 20:744–749CrossRefPubMedGoogle Scholar
  13. 13.
    Kondo Y, Ito T, Nakagami H, Hirakawa Y, Saito M, Tamaki T et al (2014) Plant GSK3 proteins regulate xylem cell differentiation downstream of TDIF-TDR signalling. Nat Commun 5:3504CrossRefGoogle Scholar
  14. 14.
    Kondo Y, Fujita T, Sugiyama M, Fukuda H (2015) A novel system for xylem cell differentiation in Arabidopsis thaliana. Mol Plant 8:612–621CrossRefPubMedGoogle Scholar
  15. 15.
    Kondo Y, Nurani AM, Saito C, Ichihashi Y, Saito M, Yamazaki K et al (2016) Vascular cell induction culture system using arabidopsis leaves (VISUAL) reveals the sequential differentiation of sieve element-like cells. Plant Cell 28:1250–1262CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Saito M, Nurani AM, Kondo Y, Fukuda H (2017) Tissue culture for xylem differentiation with arabidopsis leaves. Methods Mol Biol 1544:59–65CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biological Sciences, Graduate School of ScienceThe University of TokyoTokyoJapan

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