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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
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)
IEB ASCR
DPP Charles University
Auxin transport at the cellular level ...
IEB ASCR
DPP FS Charles
University
Actin cytoskeleton and endosomal traffickingplays a role in auxin transport ...
But !!! – clear evidence that PIN protein really transports auxin is still missing
Estelle, M.; Nature 413, 374-375, 2001
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
=>
IEB ASCR
DPP Charles University
The application of auxin efflux inhibitorsincreases auxin accumulation inside cells ...
0 5 10 15 20 25 30 35 40
Control20 M BFA
0 5 10 15 20 25 30
Control10 M NPA
0.2
0
0.4
0.6
0.8
0
0.4
0.8
1.2
1.6
2
Time (min)Time (min)
Net
3 H-N
AA
acc
um
ula
tio
n(p
mo
l .
10-6 c
ells
)
BFA NPA
Petrasek et al., Plant Physiology 131, 254-263, 2003
IEB ASCR
DPP Charles University
NPA (50M)Cortical AFs
ControlCortical AFs
ControlRadial AFs
TRITC-Phalloidin staining
BFA (20M)Cortical AFs
NPA (50M)Radial AFs
BFA (20M)Radial AFs
The application of BFA affects the structure of actin filaments ...
Petrasek et al., Plant Physiology 131, 254-263, 2003
IEB ASCR
DPP Charles University
Petrasek et al., Plant Physiology 131, 254-263, 2003
The application of BFA affect the structure of endoplasmic reticulum ...
ER-targeted GFP (mGFP5-ER) fluorescence
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
IEB ASCR
DPP Charles University
GreenPIN1-GFP
Merged
RedCalcofluor whiteCell wall
In vivo observations of AtPIN1-GFP in stable transformants of BY-2
Plasmolysis with 1M NaCl
Petrasek et al., in preparation, 2004
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
Prebleach Postbleach FRAP 30 min
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
IEB ASCR
DPP Charles University
Inducible overexpression of AtPIN7 in BY-2 cells
Petrasek et al., in preparation, 2004
Noninduced cells after 3 days
DEX-induced cells after 3 days
Starch accumulation in DEX-induced cells after 3 days
0 1 2 3 4 5 6 70
10
20
30
40
50
Cell n
um
ber
(10
4 c
ells .
ml-1
)
Time (days)
DEXTA-PIN7
DMSO TA-PIN7
DMSO Control
DEX Control
IEB ASCR
DPP Charles University
NPA prevents all changes caused by DEX-induced AtPIN7 overexpression
0 5 10 15 20 25 30 350
500
1000
1500
2000
2500
3000TA-PIN7 cells + DMSO + NPATA-PIN7 cells + DMSO (control)TA-PIN7 cells + DEXTA-PIN7 cells + DEX + NPA
Petrasek et al., in preparation, 2004
DEX-induced cells after 3 days in 10 μM NPA
Acc
um
ula
tio
n o
f 3 H
-NA
A(d
pm
. c
m-2 o
f ce
ll s
urf
ace)
IEB ASCR
DPP Charles University
Conclusion – PIN enhances auxin flow in BY-2
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.