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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 ... Plant IEB ASCR DPP Charles University Auxin flow Root PIN proteins, presumptive auxin efflux carriers AUX proteins, presumptive auxin uptake carriers Vasculature cells in protophloem files (Modified from Grebe, M.; BioEssays 26, 719-729, 2004) Auxin transport at the cellular level ... IEB ASCR DPP Charles University Actin cytoskeleton and endosomal trafficking plays a role in auxin transport ... But !!! – clear IEB ASCR evidence that PIN protein really transports auxin is still missing Estelle, M.; Nature 413, 374-375, 2001 DPP FS Charles University Why BY-2 cell line in auxin transport studies??? IEB ASCR • 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 DPP FS Charles University Net 3H-NAA accumulation (pmol . 10-6 cells) The application of auxin efflux inhibitors increases auxin accumulation inside cells ... BFA DPP Charles University NPA 2 0.8 IEB ASCR Control 20 M BFA Control 10 M NPA 1.6 0.6 1.2 0.4 0.8 0.2 0.4 0 0 0 5 10 15 20 25 30 35 40 Time (min) 0 5 10 15 20 25 30 Time (min) Petrasek et al., Plant Physiology 131, 254-263, 2003 The application of BFA affects the structure of actin filaments ... BFA (20 M) Cortical AFs Control Cortical AFs Control Radial AFs BFA (20 M) Radial AFs TRITC-Phalloidin staining IEB ASCR DPP Charles University NPA (50 M) Cortical AFs NPA (50 M) Radial AFs Petrasek et al., Plant Physiology 131, 254-263, 2003 The application of BFA affect 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 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 In vivo observations of AtPIN1-GFP in stable transformants of BY-2 Green PIN1-GFP Merged IEB ASCR DPP Charles University Red Calcofluor white Cell wall Plasmolysis with 1M NaCl Petrasek et al., in preparation, 2004 In vivo observations of AtPIN1-GFP in stable transformants of BY-2 Prebleach Postbleach IEB ASCR DPP Charles University FRAP 30 min Fluorescence recovery/redistribution after photobleaching in AtPIN1-GFP BY-2 cells Petrasek et al., in preparation, 2004 Inducible overexpression of AtPIN7 in BY-2 cells Noninduced after 24 h IEB ASCR DPP Charles University DEX-induced 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 Inducible overexpression of AtPIN7 in BY-2 cells Starch accumulation in DEX-induced cells after 3 days DPP Charles University DEX-induced cells after 3 days 50 Cell number (104 cells . ml-1) Noninduced cells after 3 days IEB ASCR 40 DMSO Control 30 DEX Control DMSO TA-PIN7 DEX TA-PIN7 20 10 0 0 1 2 3 4 5 Time (days) 6 7 Petrasek et al., in preparati on, 2004 DEX-induced cells after 3 days in 10 μM NPA Accumulation of 3H-NAA (dpm . cm-2 of cell surface) NPA prevents all changes caused by DEX-induced AtPIN7 overexpression IEB ASCR DPP Charles University 3000 TA-PIN7 cells + DMSO + NPA TA-PIN7 cells + DMSO (control) TA-PIN7 cells + DEX TA-PIN7 cells + DEX + NPA 2500 2000 1500 1000 500 0 0 5 10 15 20 25 30 35 Petrasek et al., in preparation, 2004 Conclusion – PIN enhances auxin flow in BY-2 IEB ASCR DPP Charles University 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.