Skip to main content
SpringerLink
Log in

A Forward Genetic Screen for New Regulators of Auxin-mediated Degradation of Auxin Transport Proteins in Arabidopsis thaliana

  • Published:
Journal of Plant Growth Regulation Aims and scope Submit manuscript

Abstract

The plant hormone auxin (indole-3-acetic acid) is a major regulator of plant growth and development including embryo and root patterning, lateral organ formation and growth responses to environmental stimuli. Auxin is directionally transported from cell to cell by the action of specific auxin influx [AUXIN-RESISTANT1 (AUX1)] and efflux [PIN-FORMED (PIN)] transport regulators, whose polar, subcellular localizations are aligned with the direction of the auxin flow. Auxin itself regulates its own transport by modulation of the expression and subcellular localization of the auxin transporters. Increased auxin levels promote the transcription of PIN2 and AUX1 genes as well as stabilize PIN proteins at the plasma membrane, whereas prolonged auxin exposure increases the turnover of PIN proteins and their degradation in the vacuole. In this study, we applied a forward genetic approach, to identify molecular components playing a role in the auxin-mediated degradation. We generated EMS-mutagenized Arabidopsis PIN2::PIN2:GFP, AUX1::AUX1:YFP eir1 aux1 populations and designed a screen for mutants with persistently strong fluorescent signals of the tagged PIN2 and AUX1 after prolonged treatment with the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D). This approach yielded novel auxin degradation mutants defective in trafficking and degradation of PIN2 and AUX1 proteins and established a role for auxin-mediated degradation in plant development.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Abas L, Benjamins R, Malenica N, Paciorek T, Wiśniewska J, Moulinier-Anzola JC, Sieberer T, Friml J, Luschnig C (2006) Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism. Nat Cell Biol 8:249–256

    Article  CAS  PubMed  Google Scholar 

  • Adamowski M, Friml J (2015) PIN-dependent auxin transport: action, regulation, and evolution. Plant Cell 27:20–32

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Baster P, Robert S, Kleine-Vehn J, Vanneste S, Kania U, Grunewald W, De Rybel B, Beeckman T, Friml J (2013) SCF(TIR1/AFB)-auxin signalling regulates PIN vacuolar trafficking and auxin fluxes during root gravitropism. EMBO J 32:260–274

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bennett MJ, Marchant A, Green HG, May ST, Ward SP, Millner PA, Walker AR, Schulz B, Feldmann KA (1996) Arabidopsis AUX1 gene: a permease-like regulator of root gravitropism. Science 273:948–950

    Article  CAS  PubMed  Google Scholar 

  • Dharmasiri N, Dharmasiri S, Weijers D, Lechner E, Yamada M, Hobbie L, Ehrismann SJ, Jürgens G, Estelle M (2005) plant development is regulated by a family of auxin receptor F box proteins. Dev Cell 9:109–119

    Article  CAS  PubMed  Google Scholar 

  • Feraru E, Friml J (2008) PIN polar targeting. Plant Physiol 147:1553–1559

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 22:2812–2824

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kleine-Vehn J, Dhonukshe P, Sauer M, Brewer PB, Wiśniewska J, Paciorek T, Benková E, Friml J (2008) ARF GEF-dependent transcytosis and polar delivery of PIN auxin carriers in Arabidopsis. Curr Biol 18:526–531

    Article  CAS  PubMed  Google Scholar 

  • Koizumi K, Naramoto S, Sawa S, Yahara N, Ueda T, Nakano A, Sugiyama M, Fukuda H (2005) VAN3 ARFGAP-mediated vesicle transport is involved in leaf vascular network formation. Development 132:1699–1711

    Article  CAS  PubMed  Google Scholar 

  • Laxmi A, Pan J, Morsy M, Chen R (2008) light plays an essential role in intracellular distribution of auxin efflux carrier PIN2 in Arabidopsis thaliana. PLoS ONE 3:1510

    Article  Google Scholar 

  • Löfke C, Zwiewka M, Heilmann I, Van Montagu MCE, Teichmann T, Friml J (2013) Asymmetric gibberellin signaling regulates vacuolar trafficking of PIN auxin transporters during root gravitropism. PNAS 110:3627–3632

    Article  PubMed  PubMed Central  Google Scholar 

  • Luschnig C, Gaxiola RA, Grisafi P, Fink GR (1998) EIR1, a root-specific protein involved in auxin transport, is required for gravitropism in Arabidopsis thaliana. Genes Dev 14:2175–2187

    Article  Google Scholar 

  • Malamy JE, Benfey PN (1997) Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124:33–44

    CAS  PubMed  Google Scholar 

  • Marchant A, Kargul J, May ST, Muller P, Delbarre A, Perrot-Rechenmann C, Bennett MJ (1999) AUX1 regulates root gravitropism in Arabidopsis by facilitating auxin uptake within root apical tissues. EMBO J 18:2066–2073

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Marhavy P, Bielach A, Abas L, Abuzeineh A, Duclercq J, Tanaka H, Parezová M, Petrášek J, Friml J, Kleine-Vehn J, Benková E (2011) Cytokinin modulates endocytic trafficking of PIN1 auxin efflux carrier to control plant organogenesis. Dev Cell 21:796–804

    Article  CAS  PubMed  Google Scholar 

  • Nodzynski T, Feraru MI, Hirsch S, De Rycke R, Niculaes C, Boerjan W, Van Leene J, De Jaeger G, Vanneste S, Friml J (2013) Retromer subunits VPS35A and VPS29 mediate prevacuolar compartment (PVC) function in Arabidopsis. Mol Plant 6:1849–1862

    Article  CAS  PubMed  Google Scholar 

  • Paciorek T, Zažímalová E, Ruthardt N, Petrášek J, Stierhof YD, Kleine-Vehn J, Morris DA, Emans N, Jürgens G, Geldner N, Friml J (2005) Auxin inhibits endocytosis and promotes its own efflux from cells. Nature 435:1251–1256

    Article  CAS  PubMed  Google Scholar 

  • Petrášek J, Mravec J, Bouchard R et al (2006) PIN proteins perform a rate-limiting function in cellular auxin efflux. Science 312:914–918

    Article  PubMed  Google Scholar 

  • Robert S, Kleine-Vehn J, Barbez E et al (2010) ABP1 mediates auxin inhibition of clathrin-dependent endocytosis in Arabidopsis. Cell 143:111–121

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rodríguez-Serrano M, Pazmińo DM, Sparkes I, Rochetti A, Hawes C, Romero-Puertas MC, Sandalio LM (2014) 2,4-dichlorophenoxyacetic acid promotes S-nitrosylation and oxidation of actin affecting cytoskeleton and peroxisomal dynamics. J Exp Bot 17:4783–4793

    Article  Google Scholar 

  • Sauer M, Balla J, Luschnig C, Wiśniewska J, Reinöhl V, Friml J, Benková E (2006) Canalization of auxin flow by Aux/IAA-ARF-dependentfeedback regulation of PIN polarity. Genes Dev 20:2902–2911

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Scarpella E, Marcos D, Friml J, Berleth T (2006) Control of leaf vascular patterning by polar auxin transport. Genes Dev 20:1015–1027

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Seifertová D, Skupa P, Rychtář J et al (2014) Characterization of transmembrane auxin transport in Arabidopsis suspension-cultured cells. J Plant Physiol 171:429–437

    Article  PubMed  Google Scholar 

  • Sieberer T, Seifert GJ, Hauser MT, Grisafi P, Fink GR, Luschnig C (2000) Post-transcriptional control of the Arabidopsis auxin efflux carrier EIR1 requires AXR1. Curr Biol 10:1595–1598

    Article  CAS  PubMed  Google Scholar 

  • Swarup R, Friml J, Marchant A, Ljung K, Sandberg G, Palme K, Bennett M (2001) Localization of the auxin permease AUX1 suggests two functionally distinct hormone transport pathways operate in the Arabidopsis root apex. Genes Dev 15:2648–2653

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tanaka H, Kitakura S, De Rycke R, De Groodt R, Friml J (2009) Fluorescence imaging-based screen identifies ARF GEF component of early endosomal trafficking. Curr Biol 19:391–397

    Article  CAS  PubMed  Google Scholar 

  • Tanaka H, Kitakura S, Rakusova H, Uemura T, Feraru MI, De Rycke R, Robert S, Kakimoto T, Friml J (2013) Cell polarity and patterning by PIN trafficking through early endosomal compartments in Arabidopsis thaliana. PLoS Genet 9:e1003540

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tanaka H, Nodzynski T, Kitakura S, Feraru MI, Sasabe M, Ishikawa T, Kleine-Vehn J, Kakimoto T, Friml J (2014) BEX1/ARF1A1C is required for BFA-sensitive recycling of PIN auxin transporters and auxin-mediated development in Arabidopsis. Plant Cell Physiol 55:737–749

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Vieten A, Vanneste S, Wiśniewska J, Benková E, Benjamins R, Beeckman T, Luschnig C, Friml J (2005) Functional redundancy of PIN proteins is accompanied by auxin-dependent cross-regulation of PIN expression. Development 132:4521–4531

    Article  CAS  PubMed  Google Scholar 

  • Wiśniewska J, Xu J, Seifertová D, Brewer PB, Ruzicka K, Blilou I, Rouquié D, Benková E, Scheres B, Friml J (2006) Polar PIN localization directs auxin flow in plants. Science 312:858–860

    Article  Google Scholar 

  • Xu J, Scheres B (2005) Dissection of Arabidopsis ADP-RIBOSYLATION FACTOR 1 function in epidermal cell polarity. Plant Cell 17:525–536

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang J, Vanneste S, Brewer PB et al (2011) Inositol trisphosphate-induced Ca2+ signaling modulates auxin transport and PIN polarity. Dev Cell 20:855–866

    Article  CAS  PubMed  Google Scholar 

  • Zwiewka M, Feraru E, Möller 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 21:1711–1722

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank Eva Huspekova and David John Hanley for kindly help in preparing this manuscript, and Klara Harmanova and Radka Holbova for technical help. This work was supported by the European Research Council (project ERC-2011-StG-20101109-PSDP); European Social Fund (CZ.1.07/2.3.00/20.0043) and the Czech Science Foundation GAČR (GA13-40637S) to JF. Work was realized in CEITEC – Central European Institute of Technology (CZ.1.05/1.1.00/02.0068). M.Z. was supported by Project Postdoc I (CZ.1.07/2.3.00/30.0009) co-financed by the European Social Fund and the state budget of the Czech Republic. H.S.R was supported by the SoMoProII program (3SGA5602), co-financed by the South-Moravian Region and the EU (FP/2007-2013, Grant Agreement No. 291782).

Author information

Authors and Affiliations

  1. Mendel Centre for Genomics and Proteomics of Plant Systems, CEITEC MU – Central European Institute of Technology, Masaryk University, 625 00, Brno, Czech Republic

    Radka Zemová, Marta Zwiewka, Agnieszka Bielach & Hélène S. Robert

  2. Institute of Science and Technology Austria (IST Austria), 3400, Klosterneuburg, Austria

    Jiří Friml

Authors
  1. Radka Zemová
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marta Zwiewka
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Agnieszka Bielach
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hélène S. Robert
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jiří Friml
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jiří Friml.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary material 1 (docx 1095 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zemová, R., Zwiewka, M., Bielach, A. et al. A Forward Genetic Screen for New Regulators of Auxin-mediated Degradation of Auxin Transport Proteins in Arabidopsis thaliana . J Plant Growth Regul 35, 465–476 (2016). https://doi.org/10.1007/s00344-015-9553-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00344-015-9553-2

Keywords

Search

Navigation