Download Department of Plant Physiology, Faculty of Science, Charles University

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???
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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
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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
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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
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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 ...
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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
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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
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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
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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
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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
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University
DEX-induced
cells after 3
days
50
Cell number
(104 cells . ml-1)
Noninduced
cells after 3
days
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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
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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
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DPP Charles
University
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.:
A6038303.