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

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

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

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

5. IEB ASCR DPP FS Charles University Why BY-2 cell line in auxin transport studies??? 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 Homogeneous cell population allowing easy determination of cell density and size Standard growth cycle Easy transformation and subsequent phenotyping =>=>

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

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

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

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

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

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

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

13. IEB ASCR DPP Charles University Inducible overexpression of AtPIN7 in BY-2 cells Petrasek et al., in preparati on, 2004 Noninduced cells after 3 days DEX-induced cells after 3 days Starch accumulation in DEX-induced cells after 3 days Cell number (10 4 cells. ml -1 ) Time (days) DEX TA-PIN7 DMSO TA-PIN7 DMSO Control DEX Control

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

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

16. IEB ASCR DPP Charles University Acknowledgements … 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.