1. 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

2. DPP Charles University Vasculature cells in protophloem files
Auxin (IAA) transport in plants ...
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Plant
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, , 2004)

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Auxin transport at the cellular level ...
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Actin cytoskeleton and endosomal trafficking
plays a role in auxin transport ...
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But !!! – clear evidence that PIN protein really transports auxin is still missing
Estelle, M.; Nature 413, , 2001

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Why BY-2 cell line in auxin transport studies???
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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. DPP Charles University Net 3H-NAA accumulation
The application of auxin efflux inhibitors
increases auxin accumulation inside cells ...
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BFA
NPA 2
0.8
1.6
0.6
1.2
Net 3H-NAA accumulation
(pmol cells)
0.4
0.8
0.2
0.4
Time (min)
Time (min)
Petrasek et al., Plant Physiology 131, , 2003

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

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The application of BFA affects the structure of endoplasmic reticulum ...
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ER-targeted GFP (mGFP5-ER) fluorescence
Petrasek et al., Plant Physiology 131, , 2003

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

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

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

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Inducible overexpression of AtPIN7 in BY-2 cells
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Noninduced after 24 h
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

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

14. DPP Charles University (dpm . cm-2 of cell surface)
NPA prevents all changes caused by DEX-induced AtPIN7 overexpression
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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

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Conclusion – PIN enhances auxin flow in BY-2
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Acknowledgements …
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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.: A