Intracellular trafficking and proteolysis of the
Arabidopsis auxin-efflux facilitator PIN2 are
involved in root gravitropism
Background info
Abas et al (2006). Nature Cell Biology 8:249-256
Courtney A. Hollender
CBMG 688R
PIN efflux proteins regulate auxin
movement in roots
An Introduction to auxin
Auxin
Plant growth stimulating hormone
~10-6 M optimal for shoot growth
~10-10 M for root growth
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Polarized Active transport
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AUX1 for influx
PIN proteins for efflux
Figure 19.15 of Taiz (2006) Plant Physiology
Gravitropism involves the auxin
gradients
Gravi-stimulation (laying a root on its side)
causes an auxin increase in the elongation zone of the
lower side of root
growth inhibition on lower side --> bending
BFA interferes with PIN localization
Brefeldin A (BFA) is an inhibitor of vesicular
budding
Necessary for vesicle cycling
BFA treatment of roots:
PIN2 (aka EIR1 and AGR) is involved with Auxin
distribution
Directs auxin from root tip to elongation zone
pin2 mutants
No lateral auxin gradient in roots after gravi-stimulation
Severe gravitropism deficiencies
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interferes with intracellular PIN localization
associated with gravitropic root bending deficiencies
PIN cycling is a method of post-transcriptional
regulation
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Proteosomes and Auxin affect
PIN2 concentrations
Proteasomes
Previously shown to affect PIN2 abundance
Also a post-transcriptional regulatory mechanism
Abas et al. investigated post-transcriptional
mechanisms associated with PIN2 and
gravitropism in roots
In Particular:
Long-term Auxin treatment
Proteasome degradation
Endosomal cycling
Auxin treatment
Reduces the abundance of PIN2 protein
May promote PIN2 degradation
S1. The PIN2 constructs can rescue eir1-4 (pin2) phenotype
PIN2 expression constructs
Dex inducible TA-PIN2
DMSO
They transformed PIN2 into wt and ein1-4 (pin2)
Arabidopsis mutants with the following promoters
After 8 hrs of gravi-stimulation, the
root angle for eir1-4 transformed with
35S:PIN2 plants is comparable to wt.
DEX
Heterologous/constitive promoter (OE)
CaMV 35S:PIN2 and 35S:PIN2-GFP
35S:PIN2 Auxin Localization
Dexamethasone Inducible promoter (OE)
TA:PIN2 and TA:PIN2-GFP
Endogenous promoter
PIN2:PIN2-GFP
PIN2:PIN2:HA
DR5-GFP shows eir1-4 35S:PIN2 has an asymmetric
auxin distribution similar to that of the wt
Fig. 1. The gravitropism rescuing constructs caused
polarized PIN2 expression in transverse side of
epidermal and lateral root-cap cells, similar to wt.
eir1-4
35S::PIN2
Wild type
eir1-4
PIN2:PIN2-GFP
Wt and eir1-4 seedlings treated with proteasome inhibitors MG132
and clastolactacystin β -lactone (LAC) for 150 min.
*
DEX treated
eir1-4
TA:PIN2-GFP
**
Epidermis and
lat root cap cells
of eir1-4
TA:PIN2-GFP
*eir1-4
35S:PIN2 GFP
Red = propidium iodide staining cell walls
Fig 2. PIN2 protein turnover is affected by the
proteasome and endosomal cycling
Anti-PIN for total
membrane protein
*PIN2 signal in
columella cells
PIN2 protein levels
increased when treated
with MG132 and LAC
eir1-4
35S:PIN
Anti
ubiquitin.
**Weak GFP
signal in other
parts of root
meristem
Anti
PIN2
No differences in
35S:PIN2 transcript
levels after MG132
PIN2 and
Ubiquitinated PIN2
levels increase with
MG132 treatment
Proteosome maintainence of [PIN2 ] indicates cycling between PM and endosome
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Fig 2d. Endosomal cycling helps to maintain
proper intracellular PIN2 protein
concentration
Fig 3. PIN2 expression and distribution
after gravitropism
wt
Inhibition of transport by BFA increases PIN2 levels
eir1-4 35S:PIN2-GFP
0 min
0 min
No change in
transcript
levels
90 min
240 min
360 min
360 min
Note: BFA did not inhibit proteasome, as there was no increase in
the levels of ubiquitinated proteins after treatment
Fig 3. The gravi-stimulation induced PIN2 gradient
relies on endosomal cycling and proteasome
degradation
Fig 4. A further look at the intracellular
distribution of PIN2 after gravi-stimulation
Eir1-4 35S:PIN2-GFP
240 min gravi-stimulation
DMSO
20uM BFA
(no cycling)
50uM MG132
(proteasome
Inhibition)
150 min
eir1-1 PIN2:PIN2:HA
The PIN2:HA accumulated
in the upper side of roots
delocalized from the PM
and gradually disappeared
from elongating epidermal
cells
MG132 (protesome inhibitor)prevented the de-localization
of PIN2 from the PM after gravistimulation
Fig 5. PIN2 distribution for wt and wav6-52
after 240min gravi-stimulation
Fig 5. PIN2wav6-52
wav6-52
wav6-52
Severely agravitropic PIN2 allele
Gly to Glu substitution at 541
PIN2 in wav6-52 is not
highly localized
wav6-52 has more PIN2 protein than
wt, but an equal amt of transcript
541 mutation stabilizes PIN2 (prevents
proteasome degradation)
wt
PIN2 immunolocalization
PINwav6-52 both localizes to PM and
accumulates in intracellular
vesicles
MG132
Wav6-52
wt
wt roots have an increase in
[PIN2] at cell walls of
lower side of root
This further supports the belief that wav6-52 has stabilized PIN2
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Fig 5. Root auxin distribution in wav6-52 and
eir1-4
Auxin reporter DR5rev-GFP
wav6-52
(stable PIN2)
wav6-52 had signal in columella
and lateral root cap cells up to
elongation zone
Absence of PIN2 (eir1-4) causes
auxin build-up in root cap
eir1-4
(No PIN2)
Is the stability and localization of PIN2 a
result of gravi-stimulation auxin
gradients?
Previously, extended treatment with auxin reduced PIN2 reporter
protein. (Does auxin degrade PIN2?)
10uM NAA treatment
PIN2 protein levels
decreased in both wt and
eir1-4 35S:PIN2
Transcript levels remain the
same in eir1-4 35S:PIN2
wt
Fig. 6
PIN2-HA localization in gravi-stimulated roots
treated with 10uM auxin analogue
eir1-1 PIN2:PIN2:HA
No intracellular signal in
epidermis of 90min 2,4-D
Control
Discussion and Conclusions
2,4-D
Auxin stays at PM
Intracellular signal for 2,4-D in
upper and lower epidermis after
180min
90 min
180 min
Short auxin incubation interferes with intracellular PIN:HA accum.
Longer inc. delocalize PIN2, and potentially lead to degradation
PIN2’s role in gravitropism is dependent
upon its subcellular localization and
abundance
PIN2 regulation through turnover is essential
The role of PIN proteins in the regulation of
root gravitropism
PIN cycling between PM and
endosomes controls subcellular
distribution
wav6-52 has stabilized PIN2 and is agravitropic
Some PINs are ubiquitinated and
degraded by the proteasome
BFA inhibition of vesicle transport resulted in increased
PIN2 levels and prevented a gravitropic response
In gravi-stimulated roots, PIN
localization and conc. directs
asymmetric auxin distribution
MG132 proteasome inhibition resulted in an accumulation of
ubiquitinated PIN2 and prevented gravitropic response
After gravi-stimulation PIN3
moves auxin to the lower side or
root cap, and PIN2 shuttles it to the
elongation zone
Ubiqutination of PIN signals proteasome degredation
PIN2
PIN3
aux
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The effect of Auxin on PIN2 is time
dependent
Short-term treatment interferes with intracellular
PIN2 accumulation in upper epidermis
Inhibits endocytosis?
Long-term treatment increased intracellular PIN2
accumulation
In Conclusion:
Post-transcriptional regulation of
PIN2 is necessary for its role in
gravitropism
Enhances degradation?
To prevent over-bending?
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