Identifying Novel Regulators of Vacuolar Trafficking by Combining Fluorescence Imaging-Based Forward Genetic Screening and In Vitro Pollen Germination

  • Qiang-Nan Feng
  • Yan ZhangEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1662)


Subcellular targeting of vacuolar proteins depends on cellular machinery regulating vesicular trafficking. Plant-specific vacuolar trafficking routes have been reported. However, regulators mediating these processes are obscure. By combining a fluorescence imaging-based forward genetic approach and in vitro pollen germination system, we show an efficient protocol of identifying regulators of plant-specific vacuolar trafficking routes.

Key words

Fluorescence imaging-based forward genetic screening Pollen tube EMS mutagenesis Vacuolar trafficking Arabidopsis 


  1. 1.
    Uemura T, Ueda T (2014) Plant vacuolar trafficking driven by RAB and SNARE proteins. Curr Opin Plant Biol 22:116–121CrossRefPubMedGoogle Scholar
  2. 2.
    Cui Y, Zhao Q, Gao C, Ding Y, Zeng Y, Ueda T, Nakano A, Jiang L (2014) Activation of the Rab7 GTPase by the MON1-CCZ1 complex is essential for PVC-to-vacuole trafficking and plant growth in Arabidopsis. Plant Cell 26:2080–2097CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Ebine K, Inoue T, Ito J, Ito E, Uemura T, Goh T, Abe H, Sato K, Nakano A, Ueda T (2014) Plant vacuolar trafficking occurs through distinctly regulated pathways. Curr Biol 24:1375–1382CrossRefPubMedGoogle Scholar
  4. 4.
    Singh MK, Kruger F, Beckmann H, Brumm S, Vermeer JE, Munnik T, Mayer U, Stierhof YD, Grefen C, Schumacher K, Jurgens G (2014) Protein delivery to vacuole requires SAND protein-dependent Rab GTPase conversion for MVB-vacuole fusion. Curr Biol 24:1383–1389CrossRefPubMedGoogle Scholar
  5. 5.
    Viotti C, Kruger F, Krebs M, Neubert C, Fink F, Lupanga U, Scheuring D, Boutte Y, Frescatada-Rosa M, Wolfenstetter S, Sauer N, Hillmer S, Grebe M, Schumacher K (2013) The Endoplasmic reticulum is the main membrane source for biogenesis of the lytic vacuole in Arabidopsis. Plant Cell 25:3434–3449CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Takeuchi M, Ueda T, Sato K, Abe H, Nagata T, Nakano A (2000) A dominant negative mutant of sar1 GTPase inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus in tobacco and Arabidopsis cultured cells. Plant J 23:517–525CrossRefPubMedGoogle Scholar
  7. 7.
    Sohn EJ, Kim ES, Zhao M, Kim SJ, Kim H, Kim YW, Lee YJ, Hillmer S, Sohn U, Jiang L, Hwang I (2003) Rha1, an Arabidopsis Rab5 homolog, plays a critical role in the vacuolar trafficking of soluble cargo proteins. Plant Cell 15:1057–1070CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Lee GJ, Sohn EJ, Lee MH, Hwang I (2004) The Arabidopsis Rab5 homologs Rha1 and Ara7 localize to the prevacuolar compartment. Plant Cell Physiol 45:1211–1220CrossRefPubMedGoogle Scholar
  9. 9.
    Kansup J, Tsugama D, Liu S, Takano T (2013) The Arabidopsis adaptor protein AP-3μ interacts with the G-protein β subunit AGB1 and is involved in abscisic acid regulation of germination and post-germination development. J Exp Bot 64:5611–5621CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Ninoles R, Rubio L, Garcia-Sanchez MJ, Fernandez JA, Bueso E, Alejandro S, Serrano R (2013) A dominant-negative form of Arabidopsis AP-3 β-adaptin improves intracellular pH homeostasis. Plant J 74:557–568CrossRefPubMedGoogle Scholar
  11. 11.
    Park M, Song K, Reichardt I, Kim H, Mayer U, Stierhof Y-D, Hwang I, Jürgens G (2013) Arabidopsis μ-adaptin subunit AP1M of adaptor protein complex 1 mediates late secretory and vacuolar traffic and is required for growth. Proc Natl Acad Sci U S A 110(25):10318–10323CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Wang JG, Li S, Zhao XY, Zhou LZ, Huang GQ, Feng C, Zhang Y (2013) HAPLESS13, the Arabidopsis μ1 adaptin, is essential for protein sorting at the trans-Golgi network/early endosome. Plant Physiol 162:1897–1910CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Feraru E, Paciorek T, Feraru MI, Zwiewka M, De Groodt R, De Rycke R, Kleine-Vehn J, Friml J (2010) The AP-3 β adaptin mediates the biogenesis and function of lytic vacuoles in Arabidopsis. Plant Cell 22:2812–2824CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Zwiewka M, Feraru E, Moller B, Hwang I, Feraru MI, Kleine-Vehn J, Weijers D, Friml J (2011) The AP-3 adaptor complex is required for vacuolar function in Arabidopsis. Cell Res 21:1711–1722CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Zhou LZ, Li S, Feng QN, Zhang YL, Zhao X, Zeng YL, Wang H, Jiang L, Zhang Y (2013) PROTEIN S-ACYL TRANSFERASE10 is critical for development and salt tolerance in Arabidopsis. Plant Cell 25:1093–1107CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.State Key Laboratory of Crop Biology, College of Life SciencesShandong Agricultural UniversityTai’anChina

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