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 hooks of dark-grown seedlings of Arabidopsis thaliana 0h D R 5 : : G U S E B S : : G U S 1h2h3h4h Air Ethylene Air Ethylene 1h2h4h 1h2h3h4h 1h2h3h4h 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 hooks of dark-grown seedlings of Arabidopsis thaliana

3h A 0h 3h E PIN1::PIN1:GFPPIN3::PIN3:GFPPIN4::PIN4:GFPPIN7::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

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-GFP 20h A PIN1-GFP 20h E PIN3-GFP 20h A PIN3-GFP 20h E PIN1 x PIN3 and their role in the apical hook maitenance...

Possible role of degradation of PINs in apical hooks? PIN1-GFP PIN4-GFPPIN7-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 ACC 1-NOA

MS 1-NOA ACC 1-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

ACC stimulates 1-NOA-sensitive auxin influx in BY-2 cells 48h of 10  M ACC, accumulation of 3 H 2,4-D, 1  M 1-NOA added at time 0

The effect of ACC on auxin influx is reversible with AgNo 3 and NBD in BY-2 cells 48h of 10  M ACC, accumulation of 3 H 2,4-D, AgNO 3 and NBD added at time 0

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 3 H 2,4-D

ACC has no obvious effect on auxin efflux in BY-2 cells 48h of 10  M ACC, accumulation of 3 H NAA, 1  M 1-NPA added at time 0

Active brassinosteroid has no obvious effect on auxin efflux in BY-2 cells 24h of 10  M ACC followed by 24h with added 100 nM 24-eppibrassinolide (Kohout), accumulation of 3 H NAA

ACC decreases 1-NOA-sensitive auxin influx in Arabidopsis thaliana Ler cells 48h of 10  M ACC, accumulation of 3 H 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: