Does ethylene influence auxin influx and efflux? Observation of auxin influx and efflux carriers...
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Transcript of Does ethylene influence auxin influx and efflux? Observation of auxin influx and efflux carriers...
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
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0h
DR
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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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0,8
1
1,2
1,4
0 5 10 15 20 25 30
time (min)
pmol
.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
.10-6
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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0,6
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time (min)
pmol
.10-6
cel
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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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0,6
0,8
1
1,2
1,4
0 5 10 15 20 25 30
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: