Does ethylene influence auxin influx and efflux?

Observation of auxin influx and efflux carriers during development of the apical

hook of dark-grown seedling of Arabidopsis thaliana

Ethylene- and auxin-inducible gene expression in the apical hooksof dark-grown seedlings of Arabidopsis thaliana

0h

0h

DR

5::

GU

S E

BS

::G

US

1h 2h 3h 4h

Air

Ethylene

Air

Ethylene

1h 2h 4h

1h 2h 3h 4h

1h 2h 3h 4h

3h

EBS::GUS (line 1-11) and DR5::GUS, gasing with ethylene (E; 1 ppm, 1l/h) or the air (A; 1l/h), dark-grown seedlings of Arabidopsis thaliana, 48 h after germination

- EBS::GUS expression is visible very early after the beginning of gasing with ethylene (3-4 hours). The staining is exclusively only in treated variants and is localized in the upper part of the hypocotyl with not so strong directionality (inner/outer).

- DR5::GUS expression is already from the beginning of the treatment at the inner site of the apical hook. After ethylene gasing GUS is more concentrated at the inner site of the hook.

Quantification of the DR5::GUS expression after ACC - Filip

Ethylene- and auxin-inducible gene expression in the apical hooksof dark-grown seedlings of Arabidopsis thaliana

3h A

0h

3h E

PIN1::PIN1:GFP PIN3::PIN3:GFP PIN4::PIN4:GFP PIN7::PIN7:GFP

PIN1, PIN3, PIN4 and PIN7 localization in vivo, gasing with ethylene (E; 1 ppm, 1l/h)or the air (A; 1l/h), 48h-old dark-grown seedlings of Arabidopsis thaliana

1h E

PIN2

3h E

PIN2::PIN2:GFP, gasing with ethylene (E; 1 ppm, 1l/h) or the air (A; 1l/h), 48h-old dark-grown seedlings of Arabidopsis thaliana

0h

0h

1h E

1h A

3h E

3h A

5h E

5h A

9h E

9h A

PIN1::PIN1:GFP, gasing with ethylene (E; 1 ppm, 1l/h) or the air (A; 1l/h), 48h-old dark-grown seedlings of Arabidopsis thaliana

PIN1 is deposited predominantly in cells that undergo cell division in the inner site of the hook towards cotyledons, ethylene seemingly by inducing

exaggeration of the hook keep this area without elongation...

• It seems that the presence of PIN1 in contrast to other PINs is more localized at the inner site of the hook and ethylene is keeping this localization more pronounced.

• PIN2, PIN3, PIN4 and PIN7 are not in contrast to PIN1 polarly localized in the inner site of the hook.

PIN1-GFP20h A

PIN1-GFP20h E

PIN3-GFP20h A

PIN3-GFP20h E

PIN1 x PIN3 and their role in the apical hook maitenance ...

Possible role of degradation of PINs in apical hooks?

PIN1-GFP PIN4-GFP PIN7-GFP

DR5::GFP, gasing with ethylene (E; 1 ppm, 1l/h) or the air (A; 1l/h), 48h-old dark-grown seedlings of Arabidopsis thaliana

6h A

6h E 9h E

9h A

DR5::GFP, gasing with ethylene (E; 1 ppm, 1l/h) or the air (A; 1l/h), 48h-old dark-grown seedlings of Arabidopsis thaliana

9h E

9h A6h A

6h E

AUX1::AUX1:YFP in the etiolated seedlings of Arabidopsis thaliana

MS ACC

1-NOA ACC1-NOA

MS

1-NOA ACC1-NOA

ACC

AUX1::AUX1:YFP in the etiolated seedlings of Arabidopsis thaliana

Does ethylene influence auxin influx and efflux?

Auxin influx and efflux assays in tobacco and Arabidopsis suspension cells

Accumulation of 3H-2,4-D

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1,2

1,4

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time (min)

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.10-6

cel

ls

BY-2

BY-2 ACC

BY ACC 1-NOA

ACC stimulates 1-NOA-sensitive auxin influx in BY-2 cells48h of 10 M ACC, accumulation of 3H 2,4-D, 1 M 1-NOA added at time 0

The effect of ACC on auxin influx is reversible with AgNo3 and NBD in BY-2 cells

48h of 10 M ACC, accumulation of 3H 2,4-D, AgNO3 and NBD added at time 0

Accumulation of 3H-2,4-D

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time (min)

pmol

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cells

BY-2

BY-2 + Ag+

BY-2 + NBD

BY-2 ACC

BY-2 ACC + Ag+

BY-2 ACC + NBD

Accumulation of 3H-2,4-D

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BY-2

BY-2 + BR

BY-2 + ACC

BY-2 + BR + ACC

The effect of ACC on auxin influx is reversible with active brassinosteroid in BY-2 cells

24h of 10 M ACC followed by 24h with added 100 nM 24-eppibrassinolide (Kohout), accumulation of 3H 2,4-D

Accumulation of 3H-NAA

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1,2

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time (min)

pmol

.10-6

cells

BY-2

BY-2-ACC

BY-2-ACC + NPA

ACC has no obvious effect on auxin efflux in BY-2 cells48h of 10 M ACC, accumulation of 3H NAA, 1 M 1-NPA added at time 0

Accumulation of 3H-NAA

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time (min)

pmol

.10-6

cel

ls BY-2

BY-2 + BR

BY-2 + ACC

BY-2 + BR + ACC

Active brassinosteroid has no obvious effecton auxin efflux in BY-2 cells

24h of 10 M ACC followed by 24h with added 100 nM24-eppibrassinolide (Kohout), accumulation of 3H NAA

ACC decreases 1-NOA-sensitive auxin influx in Arabidopsis thaliana Ler cells

48h of 10 M ACC, accumulation of 3H 2,4-D, 1 M 1-NOA added at time 0

Testing of auxin influx/efflux in tobacco BY-2 cells and Arabidopsis thaliana suspensions – preliminary conclusions

- ACC stimulates auxin influx in tobacco cells- the effect of ACC on influx is reversible with silver and NBD- 24-EBR reverses the effect of ACC on auxin influx- auxin efflux seems to be affected only very small, but if really this is significant (will be tested using NPA to discriminate NPA sensitive eflux) the conclusion would be that both auxin influx and efflux are affected with ACC- results from Arabidopsis cells needs further clarification using Ler x Col comparison

Testing of auxin influx/efflux in tobacco BY-2 cells and Arabidopsis thaliana suspensions - further experiments

- NPA sensitivity of NAA efflux influenced with ACC, is there possible small stimulation of efflux as well? (in progress)- 1-MCP instead of silver ions or NBD (its effect clearly proven in ACC-treated Arabidopsis plants), problems of silver and NBD is in their effects on auxin outflow stimulated by changes in osmotic conditions- concentration range of ACC applied in the accumulation test- phenotypical effects of ACC on cell growth and division, preliminary results show no obvious changes- measurements of endogenous IAA after ACC

Must be tested in BY-2:

- comparison of ACC effect in Ler and Col suspensions- 1-MCP block of ACC action on auxin influx- concentration range of ACC applied in the accumulation test- phenotypical effects of ACC on cell growth and division, preliminary results show no obvious changes, but perhaps some stimulation of division in Ler suspension is visible - measurements of endogenous IAA after ACC

Must be tested in Arabidopsis suspensions: