GREEN FLUORESCENT SEED, to Evaluate Vacuolar Trafficking in Arabidopsis Seeds

  • Tomoo Shimada
  • Kentaro Fuji
  • Takuji Ichino
  • Ooi-Kock Teh
  • Yasuko Koumoto
  • Ikuko Hara-Nishimura
Part of the Methods in Molecular Biology book series (MIMB, volume 1789)


Vacuolar trafficking plays a vital role in plant growth and development. In this chapter, we describe a powerful technique for the evaluation of vacuolar protein trafficking, which is designated as GREEN FLUORESCENT SEED. Based on vacuole-targeted green fluorescent protein in Arabidopsis seeds, this method enables the nondestructive isolation of mutant seeds defective in vacuolar trafficking and their visual characterization.

Key words

Arabidopsis thaliana Green fluorescent protein (GFP) GREEN FLUORESCENT SEED (GFS) Vacuolar sorting receptor 1 (VSR1) Vacuolar trafficking Vacuole 



This work was supported by a Grant-in-Aid for Scientific Research to I.H.-N. (15H05776) from the Japan Society for the Promotion of Science.


  1. 1.
    De Marcos Lousa C, Denecke J (2016) Lysosomal and vacuolar sorting: not so different after all! Biochem Soc Trans 44:891–897CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Robinson DG, Neuhaus JM (2016) Receptor-mediated sorting of soluble vacuolar proteins: myths, facts, and a new model. J Exp Bot 67:4435–4449CrossRefPubMedGoogle Scholar
  3. 3.
    Shimada T, Fuji K, Tamura K et al (2003) Vacuolar sorting receptor for seed storage proteins in Arabidopsis thaliana. Proc Natl Acad Sci U S A 100:16095–16100CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Fuji K, Shimada T, Takahashi H et al (2007) Arabidopsis vacuolar sorting mutants (green fluorescent seed) can be identified efficiently by secretion of vacuole-targeted green fluorescent protein in their seeds. Plant Cell 19:597–609CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Nishizawa K, Maruyama N, Satoh R et al (2003) A C-terminal sequence of soybean β-conglycinin α' subunit acts as a vacuolar sorting determinant in seed cells. Plant J 34:647–659CrossRefPubMedGoogle Scholar
  6. 6.
    Tamura K, Shimada T, Ono E et al (2003) Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants. Plant J 35:545–555CrossRefPubMedGoogle Scholar
  7. 7.
    Silady RA, Kato T, Lukowitz W et al (2004) The gravitropism defective 2 mutants of Arabidopsis are deficient in a protein implicated in endocytosis in Caenorhabditis elegans. Plant Physiol 136:3095–3103. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Tamura K, Takahashi H, Kunieda T et al (2007) Arabidopsis KAM2/GRV2 is required for proper endosome formation and functions in vacuolar sorting and determination of the embryo growth axis. Plant Cell 19:320–332CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Zhang Y, Grant BHirsh D (2001) RME-8, a conserved J-domain protein, is required for endocytosis in Caenorhabditis elegans. Mol Biol Cell 12:2011–2021CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Silady RA, Ehrhardt DW, Jackson K et al (2008) The GRV2/RME-8 protein of Arabidopsis functions in the late endocytic pathway and is required for vacuolar membrane flow. Plant J 53:29–41CrossRefPubMedGoogle Scholar
  11. 11.
    Teh OK, Hatsugai N, Tamura K et al (2015) BEACH-domain proteins act together in a cascade to mediate vacuolar protein trafficking and disease resistance in Arabidopsis. Mol Plant 8:389–398CrossRefPubMedGoogle Scholar
  12. 12.
    Cullinane AR, Schaffer AAHuizing M (2013) The BEACH is hot: a LYST of emerging roles for BEACH-domain containing proteins in human disease. Traffic 14:749–766CrossRefPubMedGoogle Scholar
  13. 13.
    Fuji K, Shirakawa M, Shimono Y et al (2016) The adaptor complex AP-4 regulates vacuolar protein sorting at the trans-Golgi network by interacting with VACUOLAR SORTING RECEPTOR1. Plant Physiol 170:211–219CrossRefPubMedGoogle Scholar
  14. 14.
    Ichino T, Fuji K, Ueda H et al (2014) GFS9/TT9 contributes to intracellular membrane trafficking and flavonoid accumulation in Arabidopsis thaliana. Plant J 80:410–423CrossRefPubMedGoogle Scholar
  15. 15.
    Kim S, Wairkar YP, Daniels RW et al (2010) The novel endosomal membrane protein Ema interacts with the class C Vps-HOPS complex to promote endosomal maturation. J Cell Biol 188:717–734CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Ebine K, Okatani Y, Uemura T et al (2008) A SNARE complex unique to seed plants is required for protein storage vacuole biogenesis and seed development of Arabidopsis thaliana. Plant Cell 20:3006–3021CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Ebine K, Inoue T, Ito J et al (2014) Plant vacuolar trafficking occurs through distinctly regulated pathways. Curr Biol 24:1375–1382CrossRefPubMedGoogle Scholar
  18. 18.
    Gao C, Zhuang X, Cui Y et al (2015) Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation. Proc Natl Acad Sci U S A 112:1886–1891CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Isono E, Katsiarimpa A, Muller IK et al (2010) The deubiquitinating enzyme AMSH3 is required for intracellular trafficking and vacuole biogenesis in Arabidopsis thaliana. Plant Cell 22:1826–1837CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Kolb C, Nagel MK, Kalinowska K et al (2015) FYVE1 is essential for vacuole biogenesis and intracellular trafficking in Arabidopsis. Plant Physiol 167:1361–1373CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Singh MK, Kruger F, Beckmann H et al (2014) Protein delivery to vacuole requires SAND protein-dependent Rab GTPase conversion for MVB-vacuole fusion. Curr Biol 24:1383–1389CrossRefPubMedGoogle Scholar
  22. 22.
    Wu X, Ebine K, Ueda T et al (2016) AtNHX5 and AtNHX6 Are Required for the Subcellular Localization of the SNARE Complex That Mediates the Trafficking of Seed Storage Proteins in Arabidopsis. PLoS One 11:e0151658CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Tomoo Shimada
    • 1
  • Kentaro Fuji
    • 1
  • Takuji Ichino
    • 1
  • Ooi-Kock Teh
    • 1
  • Yasuko Koumoto
    • 1
  • Ikuko Hara-Nishimura
    • 1
    • 2
  1. 1.Graduate School of ScienceKyoto UniversityKyotoJapan
  2. 2.Faculty of Science and EngineeringKonan UniversityKobeJapan

Personalised recommendations