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 ...

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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

=>

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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

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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 affects the structure of endoplasmic reticulum ...

ER-targeted GFP (mGFP5-ER) fluorescence

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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

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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

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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

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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

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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)

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DPP Charles University

Conclusion – PIN enhances auxin flow in BY-2

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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.