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BY-2 as a tool to study the transport of auxins

BY-2 as a tool to study the transport of auxins. Jan Petrasek. Prague, Czech Republic. Institute of Experimental Botany, Academy of Sciences of the Czech Republic. Department of Plant Physiology, Faculty of Science, Charles University. Auxin (IAA) transport in plants. IEB ASCR.

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BY-2 as a tool to study the transport of auxins

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  1. BY-2 as atoolto study the transport of auxins Jan Petrasek Prague, Czech Republic Institute of Experimental Botany, Academy of Sciences of the Czech Republic Department of Plant Physiology, Faculty of Science, Charles University

  2. Auxin (IAA) transport in plants ... IEB ASCR DPP Charles University Plant Auxin flow PIN proteins, presumptive auxin efflux carriers Root AUX proteins, presumptive auxin uptake carriers Vasculature cells in protophloem files (Modified from Grebe, M.; BioEssays 26, 719-729, 2004)

  3. Auxin transport at the cellular level ... IEB ASCR DPP Charles University

  4. Actin cytoskeleton and endosomal trafficking plays a role in auxin transport ... IEB ASCR DPP FS Charles University But !!!– clear evidence that PIN protein really transports auxin is still missing Estelle, M.; Nature 413, 374-375, 2001

  5. Why BY-2 cell line in auxin transport studies??? IEB ASCR DPP FS Charles University • Homogeneous cell population allowing easy determination of cell density and size • Standard growth cycle • Easy transformation and subsequent phenotyping => • Measurement of auxin accumulation inside cells • Visualization of cell structures involved in auxin transport (cytoskeleton, endomembranes, putative auxin-transporting proteins) • Overexpression of putative auxin efflux carriers (PIN proteins) • Application of auxin transport inhibitors

  6. The application of auxin efflux inhibitors increases auxin accumulation inside cells ... IEB ASCR DPP Charles University BFA NPA 2 0.8 1.6 0.6 1.2 Net 3H-NAA accumulation (pmol . 10-6 cells) 0.4 0.8 0.2 0.4 0 0 Time (min) Time (min) Petrasek et al., Plant Physiology 131, 254-263, 2003

  7. The application of BFA affects the structure of actin filaments ... IEB ASCR DPP Charles University Control Cortical AFs NPA (50 mM)Cortical AFs BFA (20 mM)Cortical AFs Control Radial AFs BFA (20 mM)Radial AFs NPA (50 mM)Radial AFs Petrasek et al., Plant Physiology 131, 254-263, 2003 TRITC-Phalloidin staining

  8. The application of BFA affects the structure of endoplasmic reticulum ... IEB ASCR DPP Charles University ER-targeted GFP (mGFP5-ER) fluorescence Petrasek et al., Plant Physiology 131, 254-263, 2003

  9. In vivo observations of AtPIN1-GFP in stable transformants of BY-2 IEB ASCR DPP Charles University 5 optical sections through cortical cytoplasm of AtPIN1-GFP cells Petrasek et al., in preparation, 2004

  10. In vivo observations of AtPIN1-GFP in stable transformants of BY-2 IEB ASCR DPP Charles University Red Calcofluor white Cell wall Green PIN1-GFP Merged Plasmolysis with 1M NaCl Petrasek et al., in preparation, 2004

  11. In vivo observations of AtPIN1-GFP in stable transformants of BY-2 IEB ASCR DPP Charles University Prebleach Postbleach FRAP 30 min Fluorescence recovery/redistribution after photobleaching in AtPIN1-GFP BY-2 cells Petrasek et al., in preparation, 2004

  12. Inducible overexpression of AtPIN7 in BY-2 cells IEB ASCR DPP Charles University DEX-induced after 24 h Noninduced after 24 h Indirect immunofluorescence staining of AtPIN7 protein in TA-PIN7 cells (PIN7 in pTA7002; Aoyama and Chua, 1997) Petrasek et al., in preparation, 2004

  13. Inducible overexpression of AtPIN7 in BY-2 cells IEB ASCR DPP Charles University Noninduced cells after 3 days DEX-induced cells after 3 days Starch accumulation in DEX-induced cells after 3 days DMSO Control DEX Control Cell number (104 cells . ml-1) DMSO TA-PIN7 DEX TA-PIN7 Petrasek et al., in preparation, 2004 Time (days)

  14. NPA prevents all changes caused by DEX-induced AtPIN7 overexpression IEB ASCR DPP Charles University DEX-induced cells after 3 days in 10 μM NPA Accumulation of 3H-NAA (dpm . cm-2 of cell surface) Petrasek et al., in preparation, 2004

  15. Conclusion – PIN enhances auxin flow in BY-2 IEB ASCR DPP Charles University

  16. Acknowledgements … IEB ASCR DPP Charles University • Institute of Experimental Botany (Eva Zazimalova) • Eva Zazimalova - auxin accumulation assays • Jan Petrasek - microscopy, gene transformations, auxin accumulation assays • Lucie Perry – RT-PCR, QT-PCR • Adriana Cerna - actin dynamics • Daniela Seifertova – phenotyping, PCR • Milada Covanová - technician • Department of Plant Physiology, Faculty of Science, Charles University (Zdenek Opatrny) • Katerina Schwarzerova – cytoskeleton • University Tuebingen, ZMBP (Gerd Juergens) • Jiri Friml – PIN antibodies and PIN gene constructs Supported by the Ministry of Education, Youth and Sports of the Czech Republic (project Research Centres, no.: LN00A081), and by the GAAS of the Czech Republic, project no.: A6038303.

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